Lipidomic biomarkers for the prediction of cardiovascular outcomes in coronary artery disease patients undergoing statin treatment

ABSTRACT

The present invention inter alia provides a method, and use thereof, of predicting severe CVD complications such as AMI or CVD death by detecting the lipid concentrations or lipid ratios of a biological sample and comparing it to a control and has identified specific lipid markers that are more specific and sensitive in predicting these CVD complications than currently utilized clinical markers. Also provided are antibodies towards said lipids, and the use thereof for predicting, diagnosing, preventing and/or treating CVD complications. The invention additionally relates to kits comprising lipids and/or an antibody thereto, for use in the prediction and/or diagnosis of CVD complications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage application ofPCT/EP2012/071973 filed 7 Nov. 2012, which claims priority to Europeanpatent application 11188325.2 filed 8 Nov. 2011 and U.S. ProvisionalPatent Application 61/556,915 filed 8 Nov. 2011, the entire disclosuresof which are hereby incorporated herein by reference in theirentireties.

FIELD OF THE INVENTION

This invention relates to methods and uses involving lipid levels topredict and prevent severe cardiovascular disease-associated fatalcomplications. The invention thus provides a means to identify and treathigh-risk coronary artery disease patients. The methods includeanalyzing lipid levels of a biological sample, and comparing it to acontrol.

BACKGROUND OF THE INVENTION

Worldwide, cardiovascular diseases (CVD) are among the leading causes ofmortality and morbidity with ever-increasing prevalence. CVD is used toclassify numerous conditions that affect the heart, heart valves, blood,and vasculature of the body. One of these conditions is coronary arterydisease (CAD). Statins are a family of cholesterol lowering drugs forpeople at high risk of cardiovascular complications. Statins are widelyused, as alone in the USA there are almost 20 million statin treatedpatients and it has been calculated that some 50 million patients wouldbenefit of statin treatment in the USA. However, despite statintreatment the CVD patients have risk to develop severe CVDcomplications. Early targeted initiation of preventive measures ofCVD-related fatal complications, such as acute myocardial infarction(AMI) and death, would be of great benefit and can provide a majoropportunity in reducing mortality and morbidity in patients sufferingfrom CVD. To this end, accurate identification of individuals who are atrisk of developing CVD complications is essential. However, traditionalrisk assessment fails to recognize a substantial proportion of patientsat high risk while a large proportion of individuals are classified ashaving intermediate risk, leaving patient management uncertain.Additional strategies to further refine risk assessment of high-risk CVDare therefore highly needed. To this end, the inventors have evaluatedthe role of novel lipidomic biomarkers as a prognostic tool for fatalcardiovascular events in CVD patients.

Statins are widely used drugs to prevent atherosclerotic end points inCVD patients and, therefore, a significant portion of middle agedpopulation is being treated with statins. Statins do lower efficientlyLDL-cholesterol and also many other lipids in the circulation. Thus,statin treatment is significantly affecting plasma concentrations ofmany potential lipidomic markers and therefore it is important toseparately study lipidomic biomarkers in subjects on statin treatmentand without statin treatment. It is known in the clinical practice thatconventional lipid biomarkers such as LDL-cholesterol are notinformative in statin treated patients, but these patients may stillhave a substantial residual risk of CAD complications despite statintreatment. This current invention deals with subjects who are undergoingstatin treatment at the time of the risk evaluation. A novel innovativeaspect here is that the investigators are studying risk markersseparately in patients with type 2 Diabetes (DM2). DM2 is causingnumerous metabolic alterations in the human body and, therefore, DM2 mayaffect plasma levels of lipidomic biomarkers as well. Furthermore, CVDrisk lipids in non-DM2 patients and DM2 patients may not be the same andthe prognostic accuracy can be potentially greatly improved if thesesubject groups are studied separately.

Plasma or serum total cholesterol, LDL-cholesterol or HDL-cholesterolconcentrations have been used as gold standard biomarkers for CVD/CADrisk prediction. However, a number of coronary artery disease (CAD) oracute myocardial infarction (AMI) patients have LDL-C levels within therecommended range suggesting the need for additional diagnostic measuresof the residual risk. It is evident from earlier large scale populationstudies that these measurements associate with the CAD risk and CADendpoints such as AMI or cardiovascular death. Therefore, preventivetreatment strategies have so far been addressed to lower LDL-Cconcentrations (mainly by statin treatment) and more recently alsoattempts to raise HDL-C have been made (e.g., by CETP-inhibitors). Onthe other hand, it has also been observed that one half of the AMIpatients actually do have normal LDL cholesterol levels and that thereis a substantial residual risk in statin treated patients despite aLDL-C lowering. Furthermore, recent publications have demonstrated thatplasma levels of apolipoprotein B (apoB), the main surface protein onLDL particles, and LDL-C, the amount of cholesterol in those particles,are correlated and, considered separately, as positive risk factors.Plasma levels of apolipoprotein A₁, the main surface protein on HDLparticles, and HDL-C, the amount of cholesterol in those particles, arealso correlated with each other and, considered separately, as negativerisk factors. Importantly, for a given usual apoB, lower LDL-C has beenobserved to associate with a higher risk of AMI supporting the viewthat, on average, LDL particles with low cholesterol content perparticle (small, dense LDL particles) are particularly hazardous. Thus,it seems possible that LDL-C associates directly with the more dangerousmolecules carried by LDL-particles and that LDL-C is only an indirectmeasurement of the risk. Therefore, it is of importance to search formolecules e.g., certain lipid species that are directly related withhazardous (i.e., fatal) cardiovascular events.

Lipid metabolite imbalance is a probable cause of dyslipidemia and theensuing atherosclerosis manifested in its gravest form as the vulnerableatherosclerotic plaque. Atherosclerotic plaques are complex molecularformations that contain numerous lipids. However, there are otherfactors than lipid rich plaques or LDL cholesterol that make lipids anattractive group of molecules for CVD studies. Lipids are tightlyregulated which makes Lipidomic data robust and informative on thecurrent state of the studied organism. Also, lipids are one of theculmination points of a biological system, more the true outcome thanthe predictor. Combining Lipidomic data with appropriate biobankedclinical material presents a good opportunity for biomarker discovery.Moreover, lipidomics can be used as a gauge of efficacy and safety indrug development and evolving theragnostics. Lipidomic biomarkers areprime candidates for true companion diagnostics in the CVD area andpresent many opportunities for improved translational medicine as well.

The plaque building blocks and lipoprotein components that are thoughtto traffic lipids to the site of lesion formation can now be resolvedwith Lipidomic studies correlating lipid structure and composition tofunction and thereby disease pathogenesis. The number of lipid mediatorsin the human body is overwhelming. Their identification andquantification is facilitated by the advances in mass spectrometry andlipid biochemistry, which enable the simultaneous high throughputidentification and quantification of hundreds of molecular lipid speciesin several lipid classes (Ejsing C S, et al: Global analysis of theyeast lipidome by quantitative shotgun mass spectrometry. Proc Natl AcadSci USA 2009, 106:2136-2141; Stahlman M, et al: High-throughput shotgunlipidomics by quadrupole time-of-flight mass spectrometry. J ChromatogrB Analyt Technol Biomed Life Sci 2009 Hiukka A, et al: ApoCIII-enrichedLDL in type 2 diabetes displays altered lipid composition, increasedsusceptibility for sphingomyelinase, and increased binding to biglycan.Diabetes 2009, 58:2018-2026; Linden D, et al: Liver-directedoverexpression of mitochondrial glycerol-3-phosphate acyltransferaseresults in hepatic steatosis, increased triacylglycerol secretion andreduced fatty acid oxidation. FASEB J 2006, 20:434-443.) collectivelyreferred to as the lipidome. Lipidomic studies identify lipid cellulardistribution and describe their biochemical mechanisms, interactions anddynamics. Importantly, lipidomics quantifies the exact chemicalcomposition of lipidomes (Han X, Gross R W: Global analyses of cellularlipidomes directly from crude extracts of biological samples by ESI massspectrometry: a bridge to lipidomics. J Lipid Res 2003, 44:1071-1079).

Due to both high sensitivity and selectivity of lipidomics, even thesmallest sample amounts can be analyzed today. The bulk of the lipiddata in the art today presents lipids in a sum composition format, i.e.phosphatidylcholine (PC) 34:1 (Brugger B, et al: Quantitative analysisof biological membrane lipids at the low picomole level bynano-electrospray ionization tandem mass spectrometry. Proc Natl AcadSci USA 1997, 94:2339-2344) where the molecular lipid and the attachedfatty acid tails remain unidentified. The identification of molecularlipid species, e.g., PC 16:0/18:1 (Ekroos K, et al: Charting molecularcomposition of phosphatidylcholines by fatty acid scanning and ion trapMS3 fragmentation. J Lipid Res 2003, 44:2181-2192) is the main featureof advanced lipidomics, which delivers highly resolved molecular lipidspecies rather than summed fatty acid information. For example, theinformation of the type of fatty acids and their positions of attachmentto the glycerol backbone making up the particular PC molecule isrevealed. There are conventional techniques such as thin-layerchromatography combined with gas chromatography but they not onlyrequire considerably larger sample amounts and laborious samplepreparation, but they do not deliver the molecular lipid species.Despite multiple mass spectrometry techniques capable of characterizinglipid entities, most of them are still unable to deliver reliablehigh-quality quantitative data in terms of absolute or close-to absoluteconcentrations. In the context of the present invention, electrosprayionization mass spectrometry-based lipidomics is the preferredtechnology and can utilize both shotgun and targeted lipidomics forexhaustive deciphering and precise quantification of molecularlipidomes. The superior quality and specificity of shotgun and targetedlipidomics will meet stringent regulatory standards, such as goodlaboratory practice guidelines (GLP) when set-up in the properenvironment. Using these technologies quantification of up to twothousand molecular lipids is possible even in a high throughput format.

Lipidomics is a tool for differentiating patients based on theirmolecular lipid profiles. Personalized medicine and diagnostics enabledby lipidomics will facilitate the mission of the right individualreceiving the right drug at the right time and dose. Several worksemploying analytes consisting of lipids, proteins and hydrophilicmolecules among many others have been conducted to meet the needs ofpersonalized medicine. Recently, non-hypothesis-driven metabolomicscreenings have been used to identify novel CVD biomarkers.

For example, WO2004/038381 discloses a method for metabolomicallyfacilitating the diagnosis of a disease state of a subject, or forpredicting whether a subject is predisposed to having a disease statewherein the small molecule profile from a subject is obtained andcompared to a standard small molecule profile.

WO2008/148857 discloses a method to assess the risk of cardiovasculardisease in a patient (including atherosclerosis) by isolating the HDLfraction and sub-fraction from a blood sample of the patient. Thecomponents of the HDL fraction or sub-fraction to be measured wereSphingosine-1-Phosphate (S1P), sphingomyelin (SM) and Apolipoprotein A-I(apoA-1).

WO2008/11943 further discloses markers for detecting coronary arterydisease that can indicate a patient at risk of having or developingcoronary artery disease. These include 15 “first-choice” molecules whichwere: C18:3 Cholesterol ester, C32:1 Phosphatidylcholine, Alanine, Lipid(mainly VLDL), Lysine, Hexadecanoic acid, C36:2 Phosphatidylcholine,Formate, C32:2 Phosphatidylcholine, C18:2 (Linoleic Acid), Cholesterol,C 18:2 Lysophosphatidylcholine, C36:3 Phosphatidylcholine, C34:4Phosphatidylcholine and C34:3 Phosphatidylcholine.

Furthermore, US2007/0099242 describes a method to determine if a subjectis at risk to develop, or is suffering from cardiovascular disease. Themethod involves determining a change in the amount of a biomarker in thebiological sample or HDL sub-fraction thereof, compared to a controlsample, wherein the biomarker is at least one of Apolipoprotein C-IV(“ApoC-IV”), Paraoxonase 1 (“PON-1”), Complement Factor 3 (“C3”),Apolipoprotein A-IV (“ApoA-IV”), Apolipoprotein E (“ApoE”),Apolipoprotein LI (“ApoL1”), Complement Factor C4 (“C4”), ComplementFactor C4B1 (“C4B1”), Histone H2A, Apolipoprotein C-II (“ApoC-II”),Apolipoprotein M (“ApoM”), Vitronectin, Haptoglobin-related Protein andClusterin. The document also discloses a method for detecting thepresence of one or more atherosclerotic lesions wherein a change in theamount of a biomarker in the biological sample or HDL sub-fractionthereof is detected, compared to a control sample and wherein thebiomarker is selected from PON-1, C3, C4, ApoE, ApoM and C4B1. Allbiomarkers mentioned in this document are protein or lipoproteinbiomarkers.

WO2011/063470 compares the lipid profiles of patients with coronarydisease (stable) with patients with acute coronary syndrome (ACS) havingacute chest pain, ECG changes and troponin I elevations. This comparisonrevealed lipid markers that associate with troponin I and clinicalmarkers of ACS suggesting that lipids may be used as a biomarker ofacute myocardial ischemia. However, in acute cardiovascular settingtroponin I seems to be superior marker compared to lipid profiles(Meikle et al. Plasma lipidomic analysis of stable and unstable coronaryartery disease. Arterioscler Thromb Vasc Biol. 2011 November;31(11):2723-32.) and the findings do not predict patient outcome norlong-term risk of acute myocardial ischemia or cardiovascular death.

From previous work it cannot be extrapolated that lipid analysis willyield by default a CVD biomarker predictive to the fatal outcomesassociated with CVD/CAD. There remains a need for specific markersuseful for identifying specific risk patient populations within patientsgenerally suffering from or being at risk of CVD/CAD.

The present invention identifies biomarkers of high risk CVD byabsolute, or close to absolute, quantification of defined molecularlipid species instead of profiling multiple analytes. Importantly, whilemany of the existing biomarker candidates are composite fingerprints ofmultiple factors, the lipidomics approach herein shows value already ata level of single species or ratios thereof. The present applicationdiscloses an improved lipid assay approach over those in the prior artsince it takes into account factors that affect lipid metabolism such aslipid lowering treatment (e.g., statins) and diabetes. Therefore, thepresent application provides novel personalized prediction markers.

SUMMARY OF THE INVENTION

The present invention provides novel lipidomic markers for predictingand preventing severe CVD/CAD-associated complications, including AMI,stroke and death, in CVD/CAD patients undergoing statin treatment. Thesemarkers thus provide a means to identify and treat high-risk coronaryartery disease patients. Specifically, it has been found that the lipidmolecules, lipid-lipid concentration ratios and lipid-clinicalconcentration ratios provided herein, when displaying an increased ordecreased level—as the case may be—in samples from CAD patients, areuseful lipidomic markers for the methods and uses in accordance with thepresent invention. These sensitive and specific markers werespecifically tested to display superior diagnostic and prognostic valuecompared to the current clinically-used markers predictive for CVD/CADoutcomes. In fact, the currently available biomarkers such as LDL-C orHDL-C have only very limited or no value in predicting the CVD deathrisk in CAD patients. The present invention therefore represents asignificant advantage to other markers which are currently used todiagnose and/or predict CVD and CVD complications, which include LDL-C,total plasma/serum cholesterol and Apolipoprotein B and A1. Thus, thelipidomic markers provided herein allow better diagnosis of orassessment of the risk to develop major CVD complications such as AMI orCVD death.

In accordance with the present invention, methods are inter aliadisclosed herein for determining the risk of a CVD patient undergoingstatin treatment to develop CVD complications, or for determiningwarning signs of CVD risks, (including death, myocardial infarction(MI), angina pectoris, transischemic attack (TIA) and stroke) in saidpatient.

Methods according to the invention typically comprise the steps of: a)providing a biological sample from a CAD subject; b) determining a lipidconcentration, lipid-lipid concentration ratio, or lipid-clinicalconcentration ratio or (a) corresponding profile(s) from said sample(i.e., determining information on a lipidomic marker in accordance withthe invention); and c) comparing said determined lipid concentration,lipid-lipid concentration ratio, or lipid-clinical concentration ratioor said corresponding profile(s) to the corresponding lipidconcentration, lipid-lipid concentration ratio, or lipid-clinicalconcentration ratio or the corresponding profile(s) in a control.

As mentioned above, the lipidomic marker to be compared between thesubject sample and the control (or control sample) may be one or more ofthe lipid concentration(s), lipid-lipid concentration ratio(s), orlipid-clinical concentration ratio(s) or combinations thereof, i.e., thecorresponding profile(s), as described and claimed herein. In thisregard, the control or control sample allows establishment of thelipidomic marker baseline or starting point.

The lipidomic markers of the present invention allow for prediction andprevention of fatal CVD complications. This will facilitate earlierintervention, less symptom development and suffering and decreasedmorbidity/mortality associated with CVD. Thus, the lipidomic markersdescribed and claimed herein allow for individual tailoring of drugintervention for patients being at risk to develop major CVDcomplications.

In other words, the present invention discloses diagnostic and/orpredictive lipid markers and lipid-lipid or lipid-clinical concentrationratios for use in predicting CVD complications such as AMI or CVD deathin CVD patients who are undergoing statin treatment. The invention usesthe measurement of lipid concentrations, lipid-lipid and/orlipid-clinical concentration ratios to determine the risk of saidsubject to develop CVD complications such as AMI and/or CVD death. Thesubject may have previously suffered from a cardiovascular disease eventsuch as angina pectoris, myocardial infarction or stroke.

The invention encompasses the analysis of lipid concentrations,lipid-lipid concentration ratios and/or lipid-clinical concentrationratios in samples from a subject that is undergoing treatment with oneor more statins and/or any other HMG-CoA reductase inhibitor.

Accordingly, in one aspect of the invention, a method is provided fordetermining whether a subject who is undergoing statin treatment is atrisk to develop one or more CVD complications such as AMI and/or CVDdeath, said method comprising determining in a sample from said subjectone or more lipid-lipid concentration ratio(s), wherein (an) increasedor decreased lipid-lipid concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of said subject havingan increased risk of developing one or more CVD complications, such asAMI or CVD death, wherein the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: CE 19:1 (oxCE 682.6)/CE 20:5, CE 19:1 (oxCE 682.6)/CE20:4, Cer(d18:1/16:0)/PC 18:1/20:4, Cer(d18:1/20:0)/PC 18:1/20:4, CE17:1/CE 20:5, SM (d18:1/15:0) (d18:1/14:1-OH)/SM (d18:1/23:0)(d18:1/22:1-OH), Cer(d18:1/16:0)/Cer(d18:1/24:0), CE 17:1/CE 18:3, CE18:1/CE 18:3 and CE 16:0/CE 20:4 (Table 3);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/22:0)/Cer(d18:1/24:1), CE 14:0/CE 17:1, CE 20:5/SM(d18:1/16:0) (d18:1/15:1-OH), CE 14:0/CE 15:0, CE 14:0/Cer(d18:1/20:0),Cer(d18:1/24:0)/Cer(d18:1/24:1), CE 20:4/Cer(d18:1/20:0), CE14:0/Cer(d18:1/16:0), CE 20:4/Cer(d18:1/16:0), CE 20:5/Cer(d18:1/20:0),CE 20:5/Cer(d18:1/24:1), CE 20:5/Cer(d18:1/16:0) and CE20:5/Cer(d18:1/26:1) (Table 3).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: Cer(d18:1/22:0)/Cer(d18:1/24:1), CE 14:0/CE 17:1, CE20:5/SM (d18:1/16:0) (d18:1/15:1-OH), CE 14:0/CE 15:0, CE14:0/Cer(d18:1/20:0), CE 20:4/Cer(d18:1/20:0), CE 20:5/Cer(d18:1/20:0),CE 20:5/Cer(d18:1/24:1), CE 20:5/Cer(d18:1/16:0) and CE20:5/Cer(d18:1/26:1) (Table 3).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: CE 19:1 (oxCE 682.6)/CE 20:5, CE 19:1 (oxCE 682.6)/CE20:4, Cer(d18:1/16:0)/PC 18:1/20:4 and Cer(d18:1/16:0)/Cer(d18:1/24:0)(Table 6);

and the one or more lipid-lipid concentration ratio(s) whose decrease(s)is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/Cer(d18:1/24:1) and CE 20:5/Cer(d18:1/26:1) (Table 6).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is CE20:5/Cer(d18:1/26:1) (Table 6).

In another alternative embodiment, the present invention relates to amethod for determining whether a subject who is undergoing statintreatment is at risk to develop one or more CVD complications, such asAMI and/or CVD death, comprising determining in a sample from saidsubject one or more lipid-clinical concentration ratio(s), wherein (an)increased or decreased lipid-clinical concentration ratio(s) in saidsample, when compared to a control sample, is (are) indicative of saidsubject having an increased risk of developing one or more CVDcomplications, such as AMI or CVD death, wherein the one or morelipid-clinical concentration ratio(s) whose increase(s) is (are)compared to the control is (are) selected from: CE 19:1 (oxCE 682.6)/LDLcholesterol, Cer(d18:1/16:0)/HDL cholesterol andCer(d18:1/16:0)/apolipoprotein B (Table 3);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:

-   Cer(d18:1/24:0)/supersensitive C-reactive protein,    Gb3(d18:1/18:0)/supersensitive C-reactive protein,    Cer(d18:1/18:0)/supersensitive C-reactive protein,    GlcCer(d18:1/22:0)/supersensitive C-reactive protein,    LacCer(d18:1/24:1)/supersensitive C-reactive protein, PC    16:0/18:1/supersensitive C-reactive protein,    Cer(d18:1/24:1)/supersensitive C-reactive protein,    GlcCer(d18:1/20:0)/supersensitive C-reactive protein,    Cer(d18:1/16:0)/supersensitive C-reactive protein,    GlcCer(d18:1/24:1)/supersensitive C-reactive protein,    LacCer(d18:1/24:0)/supersensitive C-reactive protein,    Gb3(d18:1/24:0)/supersensitive C-reactive protein, PC    18:0/20:3/supersensitive C-reactive protein, CE 16:0/supersensitive    C-reactive protein, SM (d18:1/18:0)/supersensitive C-reactive    protein, CE 20:3/supersensitive C-reactive protein, CE    18:1/supersensitive C-reactive protein,    Cer(d18:1/22:0)/supersensitive C-reactive protein,    LacCer(d18:1/16:0)/supersensitive C-reactive protein,    GlcCer(d18:1/16:0)/supersensitive C-reactive protein,    GlcCer(d18:1/24:0)/supersensitive C-reactive protein, SM    (d18:1/18:1)/supersensitive C-reactive protein,    GlcCer(d18:1/18:0)/supersensitive C-reactive protein, PC    O-18:0/18:2-alkyl/supersensitive C-reactive protein, SM (d18:1/16:1)    (d18:1/15:2-OH)/supersensitive C-reactive protein,    Cer(d18:1/20:0)/supersensitive C-reactive protein, CE    14:0/supersensitive C-reactive protein, SM (d18:1/23:0)    (d18:1/22:1-OH)/supersensitive C-reactive protein,    Gb3(d18:1/16:0)/supersensitive C-reactive protein, SM (d18:1/14:0)    (d18:1/13:1-OH)/supersensitive C-reactive protein, PC    16:0/16:0/supersensitive C-reactive protein, PC    18:0/18:2/supersensitive C-reactive protein, PC    18:1/20:4/supersensitive C-reactive protein, SM (d18:1/24:0)    (d18:1/23:1-OH)/supersensitive C-reactive protein, CE    18:3/supersensitive C-reactive protein,    Cer(d18:1/24:0)/supersensitive C-reactive protein, CE    18:2/supersensitive C-reactive protein,    Cer(d18:1/26:1)/supersensitive C-reactive protein, DAG    16:0/18:1/supersensitive C-reactive protein, PC    18:1/18:2/supersensitive C-reactive protein, LPC 16:0/supersensitive    C-reactive protein, CE 20:4/supersensitive C-reactive protein, PC-0    16:0/24-alkyl/CRP, PC O-16:0/18:2-alkyl/supersensitive C-reactive    protein, PC 16:0/22:6/supersensitive C-reactive protein, PC    18:0/22:6/supersensitive C-reactive protein, and CE    20:5/supersensitive C-reactive protein (Table 3).

In one particular embodiment, the one or more lipid-clinicalconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is: CE 19:1 (oxCE 682.6)/LDL cholesterol (Table 3);

In a preferred embodiment, the one or more lipid-clinical concentrationratios whose increase(s) is (are) compared to the control is:Cer(d18:1/16:0)/apolipoprotein B (Table 6);

and/or the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/supersensitive C-reactive protein, PC18:0/22:6/supersensitive C-reactive protein, CE 18:3/supersensitiveC-reactive protein, LPC 16:0/supersensitive C-reactive protein, PC-016:0/24-alkyl/CRP, PC 16:0/22:6/supersensitive C-reactive protein and CE20:5/supersensitive C-reactive protein (Table 6).

For the purposes of the invention, and particularly for lipid-clinicalconcentration ratios, an Apolipoprotein A-I measurement mayalternatively be an Apolipoprotein A-II measurement.

In another aspect of the invention, a method is provided for determiningwhether a subject undergoing statin treatment who is not suffering fromtype 2 diabetes mellitus is at risk to develop one or more CVDcomplications, such as acute myocardial infarction (AMI) and/or CVDdeath, said method comprising determining in a sample from said subjectthe concentration(s) of one or more lipid(s), wherein (an) increased ordecreased concentration(s) in said sample, when compared to a controlsample, is (are) indicative of said subject having an increased risk ofdeveloping one or more CVD complications, such as AMI or CVD death,wherein the one or more lipid(s) whose increase(s) in concentration is(are) compared to the control is (are) selected from: SM (d18:1/17:0)(d18:1/16:1-OH), Cer 18:1/24:0, PC 16:0/22:6, GluCer 18:1/24:1, and CE18:3 (Tables 4a, 7 and 9); and wherein the one or more lipid(s) whosedecrease(s) in concentration is (are) compared to the control is (are)selected from:

GluCer 18:1/18:0, Cer 18:1/16:0, Cer 18:1/24:1 and PC 18:0/18:1 (Table9).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: Cer 18:1/24:0 and GluCer 18:1/24:1 (Tables 4a, 7 and 9).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:Cer 18:1/24:0, PC 16:0/22:6, GluCer 18:1/24:1 and CE 18:3 (Table 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: GluCer 18:1/18:0, Cer18:1/16:0, Cer 18:1/24:1 and PC 18:0/18:1 (Table 9).

In a particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: Cer 18:1/24:0 and GluCer 18:1/24:1 (Table 9).

In an alternative embodiment, the present invention relates to a methodfor determining whether a subject undergoing statin treatment who is notsuffering from type 2 diabetes mellitus is at risk to develop one ormore CVD complications such as AMI and/or CVD death, comprisingdetermining in a sample from said subject one or more lipid-lipidconcentration ratio(s), wherein (an) increased or decreased lipid-lipidconcentration ratio(s) in said sample, when compared to a controlsample, is (are) indicative of said subject having an increased risk ofdeveloping one or more CVD complications, such as AMI or CVD death,wherein the one or more lipid-lipid concentration ratio(s) whoseincrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/26:1)/SM (d18:1/24:0) (d18:1/23:1-OH), CE 16:1/SM (d18:1/24:0)(d18:1/23:1-OH), PC 16:0/16:1/PC O-16:0/20:4-alkyl, PC 16:0/16:1/PC18:2/18:2, CE 16:1/CE 20:5, PC 16:0/16:1/PC 18:1/20:4, PC 18:0/18:1/PC18:2/18:2, Cer(d18:1/18:0)/PC O-16:0/20:4-alkyl, Cer(d18:1/20:0)/PC18:2/18:2, Cer(d18:1/20:0)/PC O-16:0/20:4-alkyl, CE 16:1/CE 20:4,Cer(d18:1/24:1)/PC 18:2/18:2, Cer(d18:1/24:1)/PC 18:1/20:4, CE 16:1/PC18:2/18:2, CE 16:1/CE 18:3, Cer(d18:1/18:0)/PC O-16:0/18:2-alkyl, CE17:1/CE 20:4, PC 16:0/18:1/PC 18:1/20:4, PC 16:0/18:1/PCO-16:0/20:4-alkyl, CE 16:1/PC O-16:0/20:4-alkyl, PC 18:1/18:1/PC18:1/20:4, SM (d18:1/16:1) (d18:1/15:2-OH)/SM (d18:1/24:0)(d18:1/23:1-OH), CE 18:1/CE 20:4, Cer(d18:1/18:0)/Cer(d18:1/24:0),Cer(d18:1/24:1)/PC O-16:0/18:2-alkyl, PC 18:0/18:1/PC 18:0/20:3,Cer(d18:1/26:1)/PC 16:0/22:6, CE 16:1/PC 18:1/20:4, Cer(d18:1/26:1)/PC18:0/18:2, CE 16:1/Cer(d18:1/24:0), Cer(d18:1/26:1)/PC 18:1/18:2,Cer(d18:1/26:1)/PC 16:0/18:2, PC 18:1/18:2/PC 18:2/18:2 and PC16:0/18:2/PC 18:2/18:2 (Table 4b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE14:0/CE 16:1, CE 18:3/PC 16:0/18:1, CE 14:0/PC 18:0/18:1,Cer(d18:1/24:0)/Cer(d18:1/26:1), CE 20:4/PC 18:0/18:1,Gb3(d18:1/22:0)/PC 16:0/18:1, CE 18:3/PC 18:0/18:1, CE 20:5/PC16:0/16:1, CE 20:4/Cer(d18:1/26:1), CE 18:3/PC 16:0/16:1, CE18:3/Cer(d18:1/26:1), LPC 16:0/SM (d18:1/17:0) (d18:1/16:1-OH),Gb3(d18:1/22:0)/SM (d18:1/17:0) (d18:1/16:1-OH) and CE 20:5/CE 22:2(Table 4b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from:

Cer(d18:1/26:1)/SM (d18:1/24:0) (d18:1/23:1-OH) and PC 16:0/16:1/PC18:1/20:4 (Table 7); and the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: Gb3(d18:1/22:0)/PC 16:0/18:1, CE 20:4/Cer(d18:1/26:1) andCE 20:5/CE 22:2 (Table 7).

In yet another alternative embodiment the present invention relates to amethod for determining whether a subject undergoing statin treatment whois not suffering from type 2 diabetes mellitus is at risk to develop oneor more CVD complications, such as AMI and/or CVD death, comprisingdetermining in a sample from said subject one or more lipid-clinicalconcentration ratio(s), wherein (an) increased or decreasedlipid-clinical concentration ratio(s) in said sample, when compared to acontrol sample, is (are) indicative of said subject having an increasedrisk of developing one or more CVD complications, such as AMI or CVDdeath, wherein the one or more lipid-clinical concentration ratio(s)whose increase(s) is (are) compared to the control is (are) selectedfrom: SM (d18:1/17:0) (d18:1/16:1-OH)/HDL cholesterol, CE 22:2/HDLcholesterol, SM (d18:1/17:0) (d18:1/16:1-OH)/apolipoprotein A-I, PC16:0/16:1/apolipoprotein A-I, CE 22:2/apolipoprotein A-I, PC16:0/16:1/HDL cholesterol, Cer(d18:1/26:1)/HDL cholesterol,Cer(d18:1/26:1)/apolipoprotein A-I, PC 18:0/18:1/apolipoprotein A-I, PC18:0/18:1/total cholesterol, PC 16:0/18:1/apolipoprotein A-I, PC16:0/18:1/apolipoprotein A-I, Cer(d18:1/26:1)/LDL cholesterol, PC16:0/18:1/total cholesterol, PC 18:1/18:1/apolipoprotein A-I,Cer(d18:1/26:1)/total cholesterol, CE 15:0/HDL cholesterol, PC18:1/18:1/HDL cholesterol, CE 15:0/apolipoprotein A-I, PC18:1/18:1/total cholesterol, CE 17:1/HDL cholesterol, CE17:1/apolipoprotein A-I, Cer(d18:1/26:1)/apolipoprotein B, PC16:0/18:1/LDL cholesterol, CE 22:6/HDL cholesterol and PC 16:0/18:2/LDLcholesterol (Table 4c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: SM(d18:1/24:0) (d18:1/23:1-OH)/triglycerides, CE 19:1 (oxCE682.6)/supersensitive C-reactive protein, PC 17:0/18:2/supersensitiveC-reactive protein, SM (d18:1/17:0) (d18:1/16:1-OH)/supersensitiveC-reactive protein, Gb3 (d18:1/24:1)/supersensitive C-reactive protein,Gb3 (d18:1/22:0)/supersensitive C-reactive protein, SM (d18:1/15:0)(d18:1/14:1-OH)/supersensitive C-reactive protein, SM (d18:1/23:1)(d18:1/22:2-OH)/supersensitive C-reactive protein, CE22:6/supersensitive C-reactive protein, CE 15:0/supersensitiveC-reactive protein, SM (d18:1/16:0) (d18:1/15:1-OH)/supersensitiveC-reactive protein, PC 16:0/18:2/supersensitive C-reactive protein, SM(d18:1/24:1) (d18:1/23:2-OH)/supersensitive C-reactive protein and PC18:2/18:2/supersensitive C-reactive protein (Table 4c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: SM (d18:1/17:0) (d18:1/16:1-OH)/HDL cholesterol, CE22:2/HDL cholesterol and PC 16:0/16:1/apolipoprotein A-I (Table 7); andthe one or more lipid-clinical concentration ratio(s) whose decrease(s)is (are) compared to the control is (are) selected from:Gb3(d18:1/22:0)/supersensitive C-reactive protein, CE22:6/supersensitive C-reactive protein and PC 18:2/18:2/supersensitiveC-reactive protein (Table 7).

In another aspect of the invention, a method is provided for determiningwhether a subject undergoing statin treatment who is suffering from type2 diabetes mellitus is at risk to develop one or more CVD complications,such as acute myocardial infarction (AMI) and/or CVD death, said methodcomprising determining in a sample from said subject theconcentration(s) of one or more lipid(s), wherein (an) increased ordecreased concentration(s) in said sample, when compared to a controlsample, is (are) indicative of said subject having an increased risk ofdeveloping one or more CVD complications, such as AMI or CVD death,wherein the one or more lipid(s) whose increase(s) in concentration is(are) compared to the control is (are) selected from: CE 19:1 (oxCE682.6), CE 20:0, Cer 18:1/16:0, CE 16:0, PC 16:0/18:2, Cer 18:1/20:0,and SM 18:1/24:1 (Tables 5a, 8 and 9);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PC 18:2/18:2,

CE 22:6, Cer(d18:1/24:0), PC 18:0/22:6, PC P-18:0/20:4, CE 18:1, PCO-16:0/20:4-alkyl, LPC 16:0, DAG 16:0/18:2, CE 20:4, PC 18:1/20:4, PC16:0/16:1, DAG 16:0/18:1, CE 16:1, CE 18:3, CE 20:5, Cer 18:1/18:0, CE20:3, PC 16:0/16:0, Cer 18:1/22:0, PC 18:1/18:2, CE 16:1, CE 18:1 and CE20:4 (Tables 5a, 8 and 9).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: CE 19:1 (oxCE 682.6), CE 20:0, Cer 18:1/16:0, PC 16:0/18:2, Cer18:1/20:0, and SM 18:1/24:1 (Tables 5a, 8 and 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: PC 18:2/18:2, CE 22:6,Cer(d18:1/24:0), PC 18:0/22:6, PC P-18:0/20:4, CE 18:1, PCO-16:0/20:4-alkyl, LPC 16:0, DAG 16:0/18:2, CE 20:4, PC 18:1/20:4, DAG16:0/18:1, CE 16:1, CE 20:5, Cer 18:1/18:0, PC 16:0/16:0, Cer 18:1/22:0,PC 18:1/18:2, CE 16:1, CE 18:1 and CE 20:4 (Tables 5a, 8 and 9).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:Cer 18:1/16:0, CE 16:0, PC 16:0/18:2, Cer 18:1/20:0 and SM 18:1/24:1(Table 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 20:5, Cer 18:1/18:0,CE 20:3, PC 16:0/16:0, Cer 18:1/22:0, PC 18:1/18:2, CE 16:1, CE 18:1 andCE 20:4 (Table 9).

In a particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: Cer 18:1/16:0, PC 16:0/18:2, Cer 18:1/20:0 and SM18:1/24:1 (Table 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 20:5, Cer 18:1/18:0,PC 16:0/16:0, Cer 18:1/22:0, PC 18:1/18:2, CE 16:1, CE 18:1 and CE 20:4(Table 9).

In an alternative embodiment, the present invention relates to a methodfor determining whether a subject undergoing statin treatment who issuffering from type 2 diabetes mellitus is at risk to develop one ormore CVD complications such as AMI and/or CVD death, comprisingdetermining in a sample from said subject one or more lipid-lipidconcentration ratio(s), wherein (an) increased or decreased lipid-lipidconcentration ratio(s) in said sample, when compared to a controlsample, is (are) indicative of said subject having an increased risk ofdeveloping one or more CVD complications, such as AMI or CVD death,wherein the one or more lipid-lipid concentration ratio(s) whoseincrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/16:0)/DAG 16:0/18:1, Cer(d18:1/16:0)/PC 18:0/20:3,Cer(d18:1/20:0)/PC 18:0/20:3, PC 16:0/16:0/PC 18:1/20:4, CE 15:0/CE18:3, CE 15:0/CE 16:1, PC 17:0/18:2/PC 18:1/20:4, PC 16:0/18:2/PC18:1/20:4 and PC 18:0/18:2/PC 18:1/20:4 (Table 5b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE18:1/SM (d18:1/24:1) (d18:1/23:2-OH), PC P-18:0/20:4/SM (d18:1/24:1)(d18:1/23:2-OH), CE 18:1/Cer(d18:1/16:0), CE 16:1/CE 17:1, CE 18:3/PC18:0/18:2, PC 18:0/20:3/SM (d18:1/16:0) (d18:1/15:1-OH), CE18:3/GlcCer(d18:1/16:0), CE 18:3/PC 16:0/18:2, CE 14:0/Gb3(d18:1/16:0),PC 18:1/20:4/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:3/PE 18:0/18:2, PC 18:1/20:4/PCO-18:0/18:2-alkyl, CE 20:4/SM (d18:1/24:1) (d18:1/23:2-OH), CE16:1/Gb3(d18:1/16:0), CE 20:4/PC 17:0/18:2, PC 18:1/20:4/SM (d18:1/24:1)(d18:1/23:2-OH), CE 18:3/SM (d18:1/16:1) (d18:1/15:2-OH), CE 16:1/SM(d18:1/16:0) (d18:1/15:1-OH), CE 18:3/Gb3(d18:1/22:0), CE18:3/LacCer(d18:1/16:0), CE 18:3/SM (d18:1/16:0) (d18:1/15:1-OH), PC18:1/20:4/SM (d18:1/15:0) (d18:1/14:1-OH), CE 18:3/Gb3(d18:1/16:0), CE18:3/SM (d18:1/18:0), CE 16:1/SM (d18:1/24:1) (d18:1/23:2-OH), CE16:1/Cer(d18:1/16:0), CE 20:5/LacCer(d18:1/16:0), CE 18:3/SM(d18:1/24:1) (d18:1/23:2-OH), CE 18:3/SM (d18:1/15:0) (d18:1/14:1-OH),CE 20:5/Gb3(d18:1/16:0) and CE 20:5/SM (d18:1/24:1) (d18:1/23:2-OH)(Table 5b).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: CE 18:1/SM (d18:1/24:1) (d18:1/23:2-OH), PCP-18:0/20:4/SM (d18:1/24:1) (d18:1/23:2-OH), CE 16:1/CE 17:1, CE 18:3/PC18:0/18:2, PC 18:0/20:3/SM (d18:1/16:0) (d18:1/15:1-OH), CE18:3/GlcCer(d18:1/16:0), CE 18:3/PC 16:0/18:2, CE 14:0/Gb3(d18:1/16:0),PC 18:1/20:4/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:3/PE 18:0/18:2, PC 18:1/20:4/PCO-18:0/18:2-alkyl, CE 20:4/SM (d18:1/24:1) (d18:1/23:2-OH), CE16:1/Gb3(d18:1/16:0), CE 20:4/PC 17:0/18:2, PC 18:1/20:4/SM (d18:1/24:1)(d18:1/23:2-OH), CE 18:3/SM (d18:1/16:1) (d18:1/15:2-OH), CE 16:1/SM(d18:1/16:0) (d18:1/15:1-OH), CE 18:3/Gb3(d18:1/22:0), CE18:3/LacCer(d18:1/16:0), CE 18:3/SM (d18:1/16:0) (d18:1/15:1-OH), PC18:1/20:4/SM (d18:1/15:0) (d18:1/14:1-OH), CE 18:3/Gb3(d18:1/16:0), CE18:3/SM (d18:1/18:0), CE 16:1/SM (d18:1/24:1) (d18:1/23:2-OH), CE20:5/LacCer(d18:1/16:0), CE 18:3/SM (d18:1/24:1) (d18:1/23:2-OH), CE18:3/SM (d18:1/15:0) (d18:1/14:1-OH), CE 20:5/Gb3(d18:1/16:0) and CE20:5/SM (d18:1/24:1) (d18:1/23:2-OH) (Table 5b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Cer(d18:1/16:0)/DAG 16:0/18:1, Cer(d18:1/16:0)/PC18:0/20:3, PC 17:0/18:2/PC 18:1/20:4 and PC 16:0/18:2/PC 18:1/20:4(Table 8).

In yet another alternative embodiment, the present invention relates toa method for determining whether a subject undergoing statin treatmentwho is suffering from type 2 diabetes mellitus is at risk to develop oneor more CVD complications, such as AMI and/or CVD death, comprisingdetermining in a sample from said subject one or more lipid-clinicalconcentration ratio(s), wherein (an) increased or decreasedlipid-clinical concentration ratio(s) in said sample, when compared to acontrol sample, is (are) indicative of said subject having an increasedrisk of developing one or more CVD complications, such as AMI or CVDdeath, wherein the one or more lipid-clinical concentration ratio(s)whose increase(s) is (are) compared to the control is (are) selectedfrom: CE 20:0/apolipoprotein A-I, CE 20:0/total cholesterol, SM(d18:1/24:1) (d18:1/23:2-OH)/apolipoprotein A-I, SM (d18:1/24:1)(d18:1/23:2-OH)/HDL cholesterol, SM (d18:1/24:1) (d18:1/23:2-OH)/totalcholesterol, Gb3(d18:1/16:0)/HDL cholesterol, SM (d18:1/16:0)(d18:1/15:1-OH)/apolipoprotein A-I, P C O-18:0/18:2-alkyl/apolipoproteinA-I, Gb3(d18:1/16:0)/apolipoprotein A-I, SM (d18:1/24:1)(d18:1/23:2-OH)/triglycerides, SM (d18:1/15:0)(d18:1/14:1-OH)/triglycerides, Gb3(d18:1/16:0)/triglycerides, PC17:0/18:2/triglycerides and Gb3(d18:1/16:0)/total cholesterol (Table5c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: PCP-18:0/20:4/apolipoprotein B, CE 20:4/apolipoprotein A-I, CE 14:0/HDLcholesterol, PC 18:1/20:4/HDL cholesterol, CE 20:4/total cholesterol, PC16:0/16:1/apolipoprotein B, PC 18:1/20:4/total cholesterol, CE20:4/apolipoprotein B, PC 18:0/20:3/apolipoprotein B, DAG16:0/18:1/apolipoprotein B, CE 20:3/LDL cholesterol, PC18:1/20:4/apolipoprotein B, PC 18:0/20:3/LDL cholesterol, CE 20:4/LDLcholesterol, CE 20:5/total cholesterol, PC 18:1/20:4/LDL cholesterol, PCP-16:0/18:2/supersensitive C-reactive protein, DAG16:0/18:2/supersensitive C-reactive protein , CE 19:2 (oxCE680.6)/supersensitive C-reactive protein and PCP-18:0/20:4/supersensitive C-reactive protein (Table 5c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is: CE20:0/apolipoprotein A-I (Table 8);

and the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is: DAG16:0/18:2/supersensitive C-reactive protein (Table 8).

In another aspect, the present invention relates to a method forevaluating the effectiveness of a treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject undergoingstatin treatment, said method comprising determining in a sample fromsaid subject one or more lipid-lipid concentration ratio(s), wherein(an) increased or decreased lipid-lipid concentration ratio(s) in saidsample, when compared to a control sample, is (are) indicative ofeffectiveness of said treatment, wherein the one or more lipid-lipidconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: CE 19:1 (oxCE 682.6)/CE 20:5, CE 19:1(oxCE 682.6)/CE 20:4, Cer(d18:1/16:0)/PC 18:1/20:4, Cer(d18:1/20:0)/PC18:1/20:4, CE 17:1/CE 20:5, SM (d18:1/15:0) (d18:1/14:1-OH)/SM(d18:1/23:0) (d18:1/22:1-OH), Cer(d18:1/16:0)/Cer(d18:1/24:0), CE17:1/CE 18:3, CE 18:1/CE 18:3 and CE 16:0/CE 20:4 (Table 3);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/22:0)/Cer(d18:1/24:1), CE 14:0/CE 17:1, CE 20:5/SM(d18:1/16:0) (d18:1/15:1-OH), CE 14:0/CE 15:0, CE 14:0/Cer(d18:1/20:0),Cer(d18:1/24:0)/Cer(d18:1/24:1), CE 20:4/Cer(d18:1/20:0), CE14:0/Cer(d18:1/16:0), CE 20:4/Cer(d18:1/16:0), CE 20:5/Cer(d18:1/20:0),CE 20:5/Cer(d18:1/24:1), CE 20:5/Cer(d18:1/16:0) and CE20:5/Cer(d18:1/26:1) (Table 3)

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: Cer(d18:1/22:0)/Cer(d18:1/24:1), CE 14:0/CE 17:1, CE20:5/SM (d18:1/16:0) (d18:1/15:1-OH), CE 14:0/CE 15:0, CE14:0/Cer(d18:1/20:0), CE 20:4/Cer(d18:1/20:0), CE 20:5/Cer(d18:1/20:0),CE 20:5/Cer(d18:1/24:1), CE 20:5/Cer(d18:1/16:0) and CE20:5/Cer(d18:1/26:1) (Table 3).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: E 19:1 (oxCE 682.6)/CE 20:5, CE 19:1 (oxCE 682.6)/CE20:4, Cer(d18:1/16:0)/PC 18:1/20:4 and Cer(d18:1/16:0)/Cer(d18:1/24:0)(Table 6);

and the one or more lipid-lipid concentration ratio(s) whose decrease(s)is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/Cer(d18:1/24:1) and CE 20:5/Cer(d18:1/26:1) (Table 6).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is: CE20:5/Cer(d18:1/26:1) (Table 6).

In another alternative embodiment the present invention relates tomethod for evaluating the effectiveness of a treatment of CVD and/or oneor more of its complications, such as AMI or CVD death, in a subjectundergoing statin treatment, said method comprising determining in asample from said subject one or more lipid-clinical concentrationratio(s), wherein (an) increased or decreased lipid-clinicalconcentration ratio(s) in said sample, when compared to a controlsample, is (are) indicative of effectiveness of said treatment, whereinthe one or more lipid-clinical concentration ratio(s) whose increase(s)is (are) compared to the control is (are) selected from: CE 19:1 (oxCE682.6)/LDL cholesterol, Cer(d18:1/16:0)/HDL cholesterol andCer(d18:1/16:0)/apolipoprotein B (Table 3);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:

Cer(d18:1/24:0)/supersensitive C-reactive protein,Gb3(d18:1/18:0)/supersensitive C-reactive protein,Cer(d18:1/18:0)/supersensitive C-reactive protein,GlcCer(d18:1/22:0)/supersensitive C-reactive protein,LacCer(d18:1/24:1)/supersensitive C-reactive protein, PC16:0/18:1/supersensitive C-reactive protein,Cer(d18:1/24:1)/supersensitive C-reactive protein,GlcCer(d18:1/20:0)/supersensitive C-reactive protein,Cer(d18:1/16:0)/supersensitive C-reactive protein,GlcCer(d18:1/24:1)/supersensitive C-reactive protein,LacCer(d18:1/24:0)/supersensitive C-reactive protein,Gb3(d18:1/24:0)/supersensitive C-reactive protein, PC18:0/20:3/supersensitive C-reactive protein, CE 16:0/supersensitiveC-reactive protein, SM (d18:1/18:0)/supersensitive C-reactive protein,CE 20:3/supersensitive C-reactive protein, CE 18:1/supersensitiveC-reactive protein, Cer(d18:1/22:0)/supersensitive C-reactive protein,LacCer(d18:1/16:0)/supersensitive C-reactive protein,GlcCer(d18:1/16:0)/supersensitive C-reactive protein,GlcCer(d18:1/24:0)/supersensitive C-reactive protein, SM(d18:1/18:1)/supersensitive C-reactive protein,GlcCer(d18:1/18:0)/supersensitive C-reactive protein, PCO-18:0/18:2-alkyl/supersensitive C-reactive protein, SM (d18:1/16:1)(d18:1/15:2-OH)/supersensitive C-reactive protein,Cer(d18:1/20:0)/supersensitive C-reactive protein, CE14:0/supersensitive C-reactive protein, SM (d18:1/23:0)(d18:1/22:1-OH)/supersensitive C-reactive protein,Gb3(d18:1/16:0)/supersensitive C-reactive protein, SM (d18:1/14:0)(d18:1/13:1-OH)/supersensitive C-reactive protein, PC16:0/16:0/supersensitive C-reactive protein, PC 18:0/18:2/supersensitiveC-reactive protein, PC 18:1/20:4/supersensitive C-reactive protein, SM(d18:1/24:0) (d18:1/23:1-OH)/supersensitive C-reactive protein, CE18:3/supersensitive C-reactive protein, Cer(d18:1/24:0)/supersensitiveC-reactive protein, CE 18:2/supersensitive C-reactive protein,Cer(d18:1/26:1)/supersensitive C-reactive protein, DAG16:0/18:1/supersensitive C-reactive protein, PC 18:1/18:2/supersensitiveC-reactive protein, LPC 16:0/supersensitive C-reactive protein, CE20:4/supersensitive C-reactive protein, PC-0 16:0/24-alkyl/CRP, PCO-16:0/18:2-alkyl/supersensitive C-reactive protein, PC16:0/22:6/supersensitive C-reactive protein, PC 18:0/22:6/supersensitiveC-reactive protein and CE 20:5/supersensitive C-reactive protein (Table3).

In one particular embodiment, the one or more lipid-clinicalconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is: CE 19:1 (oxCE 682.6)/LDL cholesterol (Table 3).

In a preferred embodiment, the one or more lipid-clinical concentrationratios whose increase(s) is (are) compared to the control is:Cer(d18:1/16:0)/apolipoprotein B (Table 6);

and/or the one or more lipid-clinical concentration ratio(s) whosedecrease(s) in is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/supersensitive C-reactive protein, PC18:0/22:6/supersensitive C-reactive protein, CE 18:3/supersensitiveC-reactive protein, LPC 16:0/supersensitive C-reactive protein, PC-016:0/24-alkyl/CRP, PC 16:0/22:6/supersensitive C-reactive protein and CE20:5/supersensitive C-reactive protein (Table 6).

For the purposes of the invention, and particularly for lipid-clinicalconcentration ratios, an Apolipoprotein A-I measurement mayalternatively be an Apolipoprotein A-II measurement.

In another aspect the present invention relates to a method forevaluating the effectiveness of a treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject undergoingstatin treatment and not suffering from type 2 diabetes mellitus saidmethod comprising determining in a sample from said subject theconcentration(s) of one or more lipid(s), wherein (an) increased ordecreased concentration(s) in said sample, when compared to a controlsample, is (are) indicative of effectiveness of said treatment, whereinthe one or more lipid(s) whose increase(s) in concentration is (are)compared to the control is (are) selected from: SM (d18:1/17:0)(d18:1/16:1-OH), Cer 18:1/24:0, PC 16:0/22:6, GluCer 18:1/24:1 and CE18:3 (Tables 4a, 7 and 9);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from:

GluCer 18:1/18:0, Cer 18:1/16:0, Cer 18:1/24:1 and PC 18:0/18:1 (Table9).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: Cer 18:1/24:0 and GluCer 18:1/24:1 (Tables 4a, 7 and 9)

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:Cer 18:1/24:0, PC 16:0/22:6, GluCer 18:1/24:1 and CE 18:3 (Table 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: GluCer 18:1/18:0, Cer18:1/16:0, Cer 18:1/24:1 and PC 18:0/18:1 (Table 9).

In a particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: Cer 18:1/24:0 and GluCer 18:1/24:1 (Table 9).

In an alternative embodiment, the present invention relates to a methodfor evaluating the effectiveness of a treatment of CVD and/or one ormore of its complications, such as AMI or CVD death, in a subjectundergoing statin treatment and not suffering from type 2 diabetesmellitus said method comprising determining in a sample from saidsubject one or more lipid-lipid concentration ratio(s), wherein (an)increased or decreased lipid-lipid concentration ratio(s) in saidsample, when compared to a control sample, is (are) indicative ofeffectiveness of said treatment, wherein the one or more lipid-lipidconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: Cer(d18:1/26:1)/SM (d18:1/24:0)(d18:1/23:1-OH), CE 16:1/SM (d18:1/24:0) (d18:1/23:1-OH), PC16:0/16:1/PC O-16:0/20:4-alkyl, PC 16:0/16:1/PC 18:2/18:2, CE 16:1/CE20:5, PC 16:0/16:1/PC 18:1/20:4, PC 18:0/18:1/PC 18:2/18:2,Cer(d18:1/18:0)/PC O-16:0/20:4-alkyl, Cer(d18:1/20:0)/PC 18:2/18:2,Cer(d18:1/20:0)/PC O-16:0/20:4-alkyl, CE 16:1/CE 20:4,Cer(d18:1/24:1)/PC 18:2/18:2, Cer(d18:1/24:1)/PC 18:1/20:4, CE 16:1/PC18:2/18:2, CE 16:1/CE 18:3, Cer(d18:1/18:0)/PC O-16:0/18:2-alkyl, CE17:1/CE 20:4, PC 16:0/18:1/PC 18:1/20:4, PC 16:0/18:1/PCO-16:0/20:4-alkyl, CE 16:1/PC O-16:0/20:4-alkyl, PC 18:1/18:1/PC18:1/20:4, SM (d18:1/16:1) (d18:1/15:2-OH)/SM (d18:1/24:0)(d18:1/23:1-OH), CE 18:1/CE 20:4, Cer(d18:1/18:0)/Cer(d18:1/24:0),Cer(d18:1/24:1)/PC O-16:0/18:2-alkyl, PC 18:0/18:1/PC 18:0/20:3,Cer(d18:1/26:1)/PC 16:0/22:6, CE 16:1/PC 18:1/20:4, Cer(d18:1/26:1)/PC18:0/18:2, CE 16:1/Cer(d18:1/24:0), Cer(d18:1/26:1)/PC 18:1/18:2,Cer(d18:1/26:1)/PC 16:0/18:2, PC 18:1/18:2/PC 18:2/18:2 and PC16:0/18:2/PC 18:2/18:2 (Table 4b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE14:0/CE 16:1, CE 18:3/PC 16:0/18:1, CE 14:0/PC 18:0/18:1,Cer(d18:1/24:0)/Cer(d18:1/26:1), CE 20:4/PC 18:0/18:1,Gb3(d18:1/22:0)/PC 16:0/18:1, CE 18:3/PC 18:0/18:1, CE 20:5/PC16:0/16:1, CE 20:4/Cer(d18:1/26:1), CE 18:3/PC 16:0/16:1, CE18:3/Cer(d18:1/26:1), LPC 16:0/SM (d18:1/17:0) (d18:1/16:1-OH),Gb3(d18:1/22:0)/SM (d18:1/17:0) (d18:1/16:1-OH) and CE 20:5/CE 22:2(Table 4b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from:

Cer(d18:1/26:1)/SM (d18:1/24:0) (d18:1/23:1-OH) and PC 16:0/16:1/PC18:1/20:4 (Table 7); and the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: Gb3(d18:1/22:0)/PC 16:0/18:1, CE 20:4/Cer(d18:1/26:1) andCE 20:5/CE 22:2 (Table 7).

In yet another alternative embodiment, a method is provided forevaluating the effectiveness of a treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject undergoingstatin treatment and not suffering from type 2 diabetes mellitus, saidmethod comprising determining in a sample from said subject one or morelipid-clinical concentration ratio(s), wherein (an) increased ordecreased lipid-clinical concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of effectiveness ofsaid treatment, wherein the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: SM (d18:1/17:0) (d18:1/16:1-OH)/HDL cholesterol, CE22:2/HDL cholesterol, SM (d18:1/17:0) (d18:1/16:1-OH)/apolipoproteinA-I, PC 16:0/16:1/apolipoprotein A-I, CE 22:2/apolipoprotein A-I, PC16:0/16:1/HDL cholesterol, Cer(d18:1/26:1)/HDL cholesterol,Cer(d18:1/26:1)/apolipoprotein A-I, PC 18:0/18:1/apolipoprotein A-I, PC18:0/18:1/total cholesterol, PC 16:0/18:1/apolipoprotein A-I, PC16:0/18:1/apolipoprotein A-I, Cer(d18:1/26:1)/LDL cholesterol, PC16:0/18:1/total cholesterol, PC 18:1/18:1/apolipoprotein A-I,Cer(d18:1/26:1)/total cholesterol, CE 15:0/HDL cholesterol, PC18:1/18:1/HDL cholesterol, CE 15:0/apolipoprotein A-I, PC18:1/18:1/total cholesterol, CE 17:1/HDL cholesterol, CE17:1/apolipoprotein A-I, Cer(d18:1/26:1)/apolipoprotein B, PC16:0/18:1/LDL cholesterol, CE 22:6/HDL cholesterol and PC 16:0/18:2/LDLcholesterol (Table 4c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: SM(d18:1/24:0) (d18:1/23:1-OH)/triglycerides, CE 19:1 (oxCE682.6)/supersensitive C-reactive protein, PC 17:0/18:2/supersensitiveC-reactive protein, SM (d18:1/17:0) (d18:1/16:1-OH)/supersensitiveC-reactive protein, Gb3 (d18:1/24:1)/supersensitive C-reactive protein,Gb3 (d18:1/22:0)/supersensitive C-reactive protein, SM (d18:1/15:0)(d18:1/14:1-OH)/supersensitive C-reactive protein, SM (d18:1/23:1)(d18:1/22:2-OH)/supersensitive C-reactive protein, CE22:6/supersensitive C-reactive protein, CE 15:0/supersensitiveC-reactive protein, SM (d18:1/16:0) (d18:1/15:1-OH)/supersensitiveC-reactive protein, PC 16:0/18:2/supersensitive C-reactive protein, SM(d18:1/24:1) (d18:1/23:2-OH)/supersensitive C-reactive protein and PC18:2/18:2/supersensitive C-reactive protein (Table 4c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: SM (d18:1/17:0) (d18:1/16:1-OH)/HDL cholesterol, CE22:2/HDL cholesterol and PC 16:0/16:1/apolipoprotein A-I (Table 7);

and the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Gb3(d18:1/22:0)/supersensitive C-reactive protein, CE22:6/supersensitive C-reactive protein and PC 18:2/18:2/supersensitiveC-reactive protein (Table 7).

In another aspect, the present invention relates to a method forevaluating the effectiveness of a treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject undergoingstatin treatment and suffering from type 2 diabetes mellitus said methodcomprising determining in a sample from said subject theconcentration(s) of one or more lipid(s), wherein (an) increased ordecreased concentration(s) in said sample, when compared to a controlsample, is (are) indicative of effectiveness of said treatment, whereinthe one or more lipid(s) whose increase(s) in concentration is (are)compared to the control is (are) selected from: CE 19:1 (oxCE 682.6), CE20:0, Cer 18:1/16:0, CE 16:0, PC 16:0/18:2, Cer 18:1/20:0, and SM18:1/24:1 (Tables 5a, 8 and 9);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PC 18:2/18:2,

CE 22:6, Cer(d18:1/24:0), PC 18:0/22:6, PC P-18:0/20:4, CE 18:1, PCO-16:0/20:4-alkyl, LPC 16:0, DAG 16:0/18:2, CE 20:4, PC 18:1/20:4, PC16:0/16:1, DAG 16:0/18:1, CE 16:1, CE 18:3, CE 20:5, Cer 18:1/18:0, CE20:3, PC 16:0/16:0, Cer 18:1/22:0, PC 18:1/18:2, CE 16:1, CE 18:1 and CE20:4 (Tables 5a, 8 and 9).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: CE 19:1 (oxCE 682.6), CE 20:0, Cer 18:1/16:0, PC 16:0/18:2, Cer18:1/20:0, and SM 18:1/24:1 (Tables 5a, 8 and 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: PC 18:2/18:2, CE 22:6,Cer(d18:1/24:0), PC 18:0/22:6, PC P-18:0/20:4, CE 18:1, PCO-16:0/20:4-alkyl, LPC 16:0, DAG 16:0/18:2, CE 20:4, PC 18:1/20:4, DAG16:0/18:1, CE 16:1, CE 20:5, Cer 18:1/18:0, PC 16:0/16:0, Cer 18:1/22:0,PC 18:1/18:2, CE 16:1, CE 18:1 and CE 20:4 (Tables 5a, 8 and 9).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:Cer 18:1/16:0, CE 16:0, PC 16:0/18:2, Cer 18:1/20:0 and SM 18:1/24:1(Table 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 20:5, Cer 18:1/18:0,CE 20:3, PC 16:0/16:0, Cer 18:1/22:0, PC 18:1/18:2, CE 16:1, CE 18:1 andCE 20:4 (Table 9).

In a particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: Cer 18:1/16:0, PC 16:0/18:2, Cer 18:1/20:0 and SM18:1/24:1 (Table 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 20:5, Cer 18:1/18:0,PC 16:0/16:0, Cer 18:1/22:0, PC 18:1/18:2, CE 16:1, CE 18:1 and CE 20:4(Table 9).

In an alternative embodiment, the present invention relates to a methodfor evaluating the effectiveness of a treatment of CVD and/or one ormore of its complications, such as AMI or CVD death, in a subjectundergoing statin treatment and suffering from type 2 diabetes mellitussaid method comprising determining in a sample from said subject one ormore lipid-lipid concentration ratio(s), wherein (an) increased ordecreased lipid-lipid concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of effectiveness ofsaid treatment, wherein the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Cer(d18:1/16:0)/DAG 16:0/18:1, Cer(d18:1/16:0)/PC18:0/20:3, Cer(d18:1/20:0)/PC 18:0/20:3, PC 16:0/16:0/PC 18:1/20:4, CE15:0/CE 18:3, CE 15:0/CE 16:1, PC 17:0/18:2/PC 18:1/20:4, PC16:0/18:2/PC 18:1/20:4 and PC 18:0/18:2/PC 18:1/20:4 (Table 5b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE18:1/SM (d18:1/24:1) (d18:1/23:2-OH), PC P-18:0/20:4/SM (d18:1/24:1)(d18:1/23:2-OH), CE 18:1/Cer(d18:1/16:0), CE 16:1/CE 17:1, CE 18:3/PC18:0/18:2, PC 18:0/20:3/SM (d18:1/16:0) (d18:1/15:1-OH), CE18:3/GlcCer(d18:1/16:0), CE 18:3/PC 16:0/18:2, CE 14:0/Gb3(d18:1/16:0),PC 18:1/20:4/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:3/PE 18:0/18:2, PC 18:1/20:4/PCO-18:0/18:2-alkyl, CE 20:4/SM (d18:1/24:1) (d18:1/23:2-OH), CE16:1/Gb3(d18:1/16:0), CE 20:4/PC 17:0/18:2, PC 18:1/20:4/SM (d18:1/24:1)(d18:1/23:2-OH), CE 18:3/SM (d18:1/16:1) (d18:1/15:2-OH), CE 16:1/SM(d18:1/16:0) (d18:1/15:1-OH), CE 18:3/Gb3(d18:1/22:0), CE18:3/LacCer(d18:1/16:0), CE 18:3/SM (d18:1/16:0) (d18:1/15:1-OH), PC18:1/20:4/SM (d18:1/15:0) (d18:1/14:1-OH), CE 18:3/Gb3(d18:1/16:0), CE18:3/SM (d18:1/18:0), CE 16:1/SM (d18:1/24:1) (d18:1/23:2-OH), CE16:1/Cer(d18:1/16:0), CE 20:5/LacCer(d18:1/16:0), CE 18:3/SM(d18:1/24:1) (d18:1/23:2-OH), CE 18:3/SM (d18:1/15:0) (d18:1/14:1-OH),CE 20:5/Gb3(d18:1/16:0) and CE 20:5/SM (d18:1/24:1) (d18:1/23:2-OH)(Table 5b).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: CE 18:1/SM (d18:1/24:1) (d18:1/23:2-OH), PCP-18:0/20:4/SM (d18:1/24:1) (d18:1/23:2-OH), CE 16:1/CE 17:1, CE 18:3/PC18:0/18:2, PC 18:0/20:3/SM (d18:1/16:0) (d18:1/15:1-OH), CE18:3/GlcCer(d18:1/16:0), CE 18:3/PC 16:0/18:2, CE 14:0/Gb3(d18:1/16:0),PC 18:1/20:4/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:3/PE 18:0/18:2, PC 18:1/20:4/PCO-18:0/18:2-alkyl, CE 20:4/SM (d18:1/24:1) (d18:1/23:2-OH), CE16:1/Gb3(d18:1/16:0), CE 20:4/PC 17:0/18:2, PC 18:1/20:4/SM (d18:1/24:1)(d18:1/23:2-OH), CE 18:3/SM (d18:1/16:1) (d18:1/15:2-OH), CE 16:1/SM(d18:1/16:0) (d18:1/15:1-OH), CE 18:3/Gb3(d18:1/22:0), CE18:3/LacCer(d18:1/16:0), CE 18:3/SM (d18:1/16:0) (d18:1/15:1-OH), PC18:1/20:4/SM (d18:1/15:0) (d18:1/14:1-OH), CE 18:3/Gb3(d18:1/16:0), CE18:3/SM (d18:1/18:0), CE 16:1/SM (d18:1/24:1) (d18:1/23:2-OH), CE20:5/LacCer(d18:1/16:0), CE 18:3/SM (d18:1/24:1) (d18:1/23:2-OH), CE18:3/SM (d18:1/15:0) (d18:1/14:1-OH), CE 20:5/Gb3(d18:1/16:0) and CE20:5/SM (d18:1/24:1) (d18:1/23:2-OH) (Table 5b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Cer(d18:1/16:0)/DAG 16:0/18:1, Cer(d18:1/16:0)/PC18:0/20:3, PC 17:0/18:2/PC 18:1/20:4 and PC 16:0/18:2/PC 18:1/20:4(Table 8).

In yet another alternative embodiment, a method is provided forevaluating the effectiveness of a treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject undergoingstatin treatment and suffering from type 2 diabetes mellitus, saidmethod comprising determining in a sample from said subject one or morelipid-clinical concentration ratio(s), wherein (an) increased ordecreased lipid-clinical concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of effectiveness ofsaid treatment, wherein the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: CE 20:0/apolipoprotein A-I, CE 20:0/total cholesterol, SM(d18:1/24:1) (d18:1/23:2-OH)/apolipoprotein A-I, SM (d18:1/24:1)(d18:1/23:2-OH)/HDL cholesterol, SM (d18:1/24:1) (d18:1/23:2-OH)/totalcholesterol, Gb3(d18:1/16:0)/HDL cholesterol, SM (d18:1/16:0)(d18:1/15:1-OH)/apolipoprotein A-I, P C O-18:0/18:2-alkyl/apolipoproteinA-I, Gb3(d18:1/16:0)/apolipoprotein A-I, SM (d18:1/24:1)(d18:1/23:2-OH)/triglycerides, SM (d18:1/15:0)(d18:1/14:1-OH)/triglycerides, Gb3(d18:1/16:0)/triglycerides, PC17:0/18:2/triglycerides and Gb3(d18:1/16:0)/total cholesterol (Table5c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: PCP-18:0/20:4/apolipoprotein B, CE 20:4/apolipoprotein A-I, CE 14:0/HDLcholesterol, PC 18:1/20:4/HDL cholesterol, CE 20:4/total cholesterol, PC16:0/16:1/apolipoprotein B, PC 18:1/20:4/total cholesterol, CE20:4/apolipoprotein B, PC 18:0/20:3/apolipoprotein B, DAG16:0/18:1/apolipoprotein B, CE 20:3/LDL cholesterol, PC18:1/20:4/apolipoprotein B, PC 18:0/20:3/LDL cholesterol, CE 20:4/LDLcholesterol, CE 20:5/total cholesterol, PC 18:1/20:4/LDL cholesterol, PCP-16:0/18:2/supersensitive C-reactive protein, DAG16:0/18:2/supersensitive C-reactive protein, CE 19:2 (oxCE680.6)/supersensitive C-reactive protein and PCP-18:0/20:4/supersensitive C-reactive protein (Table 5c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is: CE20:0/apolipoprotein A-I (Table 8);

and the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is: DAG16:0/18:2/supersensitive C-reactive protein (Table 8).

In yet another aspect the present invention relates to a method ofchoosing an appropriate treatment of CVD and/or one or more of itscomplications, such as AMI or CVD death, in a subject undergoing statintreatment, said method comprising determining in a sample from saidsubject one or more lipid-lipid concentration ratio(s), wherein (an)increased or decreased lipid-lipid concentration ratio(s) in saidsample, when compared to a control sample, is (are) indicative of saidsubject being in need of treatment or a change in, or supplementationof, an already administered treatment, wherein the one or morelipid-lipid concentration ratio(s) whose increase(s) is (are) comparedto the control is (are) selected from: CE 19:1 (oxCE 682.6)/CE 20:5, CE19:1 (oxCE 682.6)/CE 20:4, Cer(d18:1/16:0)/PC 18:1/20:4,Cer(d18:1/20:0)/PC 18:1/20:4, CE 17:1/CE 20:5, SM (d18:1/15:0)(d18:1/14:1-OH)/SM (d18:1/23:0) (d18:1/22:1-OH),Cer(d18:1/16:0)/Cer(d18:1/24:0), CE 17:1/CE 18:3, CE 18:1/CE 18:3 and CE16:0/CE 20:4 (Table 3);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Cer(d18:1/22:0)/Cer(d18:1/24:1), CE 14:0/CE 17:1, CE 20:5/SM(d18:1/16:0) (d18:1/15:1-OH), CE 14:0/CE 15:0, CE 14:0/Cer(d18:1/20:0),Cer(d18:1/24:0)/Cer(d18:1/24:1), CE 20:4/Cer(d18:1/20:0), CE14:0/Cer(d18:1/16:0), CE 20:4/Cer(d18:1/16:0), CE 20:5/Cer(d18:1/20:0),CE 20:5/Cer(d18:1/24:1), CE 20:5/Cer(d18:1/16:0) and CE20:5/Cer(d18:1/26:1) (Table 3).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: Cer(d18:1/22:0)/Cer(d18:1/24:1), CE 14:0/CE 17:1, CE20:5/SM (d18:1/16:0) (d18:1/15:1-OH), CE 14:0/CE 15:0, CE14:0/Cer(d18:1/20:0), CE 20:4/Cer(d18:1/20:0), CE 20:5/Cer(d18:1/20:0),CE 20:5/Cer(d18:1/24:1), CE 20:5/Cer(d18:1/16:0) and CE20:5/Cer(d18:1/26:1) (Table 3).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: CE 19:1 (oxCE 682.6)/CE 20:5, CE 19:1 (oxCE 682.6)/CE20:4, Cer(d18:1/16:0)/PC 18:1/20:4 and Cer(d18:1/16:0)/Cer(d18:1/24:0)(Table 6);

and the one or more lipid-lipid concentration ratio(s) whose decrease(s)is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/Cer(d18:1/24:1) and

CE 20:5/Cer(d18:1/26:1) (Table 6).

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is: CE20:5/Cer(d18:1/26:1) (Table 6).

In another alternative embodiment, the present invention relates to amethod of choosing an appropriate treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject undergoingstatin treatment, said method comprising determining in a sample fromsaid subject one or more lipid-clinical concentration ratio(s), wherein(an) increased or decreased lipid-clinical concentration ratio(s) insaid sample, when compared to a control sample, is (are) indicative ofsaid subject being in need of treatment or a change in, orsupplementation of, an already administered treatment, wherein the oneor more lipid-clinical concentration ratio(s) whose increase(s) is (are)compared to the control is (are) selected from: CE 19:1 (oxCE 682.6)/LDLcholesterol, Cer(d18:1/16:0)/HDL cholesterol andCer(d18:1/16:0)/apolipoprotein B (Table 3);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:

Cer(d18:1/24:0)/supersensitive C-reactive protein,Gb3(d18:1/18:0)/supersensitive C-reactive protein,Cer(d18:1/18:0)/supersensitive C-reactive protein,GlcCer(d18:1/22:0)/supersensitive C-reactive protein,LacCer(d18:1/24:1)/supersensitive C-reactive protein, PC16:0/18:1/supersensitive C-reactive protein,Cer(d18:1/24:1)/supersensitive C-reactive protein,GlcCer(d18:1/20:0)/supersensitive C-reactive protein,Cer(d18:1/16:0)/supersensitive C-reactive protein,GlcCer(d18:1/24:1)/supersensitive C-reactive protein,LacCer(d18:1/24:0)/supersensitive C-reactive protein, Gb3(d18:1/24:0)/supersensitive C-reactive protein, PC18:0/20:3/supersensitive C-reactive protein, CE 16:0/supersensitiveC-reactive protein, SM (d18:1/18:0)/supersensitive C-reactive protein,CE 20:3/supersensitive C-reactive protein, CE 18:1/supersensitiveC-reactive protein, Cer(d18:1/22:0)/supersensitive C-reactive protein,LacCer(d18:1/16:0)/supersensitive C-reactive protein,GlcCer(d18:1/16:0)/supersensitive C-reactive protein,GlcCer(d18:1/24:0)/supersensitive C-reactive protein, SM(d18:1/18:1)/supersensitive C-reactive protein,GlcCer(d18:1/18:0)/supersensitive C-reactive protein, PCO-18:0/18:2-alkyl/supersensitive C-reactive protein, SM (d18:1/16:1)(d18:1/15:2-OH)/supersensitive C-reactive protein,Cer(d18:1/20:0)/supersensitive C-reactive protein, CE14:0/supersensitive C-reactive protein, SM (d18:1/23:0)(d18:1/22:1-OH)/supersensitive C-reactive protein, Gb3(d18:1/16:0)/supersensitive C-reactive protein, SM (d18:1/14:0)(d18:1/13:1-OH)/supersensitive C-reactive protein, PC16:0/16:0/supersensitive C-reactive protein, PC 18:0/18:2/supersensitiveC-reactive protein, PC 18:1/20:4/supersensitive C-reactive protein, SM(d18:1/24:0) (d18:1/23:1-OH)/supersensitive C-reactive protein, CE18:3/supersensitive C-reactive protein, Cer(d18:1/24:0)/supersensitiveC-reactive protein, CE 18:2/supersensitive C-reactive protein,Cer(d18:1/26:1)/supersensitive C-reactive protein, DAG16:0/18:1/supersensitive C-reactive protein, PC 18:1/18:2/supersensitiveC-reactive protein, LPC 16:0/supersensitive C-reactive protein, CE20:4/supersensitive C-reactive protein, PC-0 16:0/24-alkyl/CRP, PCO-16:0/18:2-alkyl/supersensitive C-reactive protein, PC16:0/22:6/supersensitive C-reactive protein, PC 18:0/22:6/supersensitiveC-reactive protein and CE 20:5/supersensitive C-reactive protein (Table3).

In one particular embodiment, the one or more lipid-clinicalconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is: CE 19:1 (oxCE 682.6)/LDL cholesterol (Table 3).

In a preferred embodiment, the one or more lipid-clinical concentrationratios whose increase(s) is (are) compared to the control is:Cer(d18:1/16:0)/apolipoprotein B (Table 6);

and/or the one or more lipid-clinical concentration ratio(s) whosedecrease(s) in is (are) compared to the control is (are) selected from:Cer(d18:1/24:0)/supersensitive C-reactive protein, PC18:0/22:6/supersensitive C-reactive protein, CE 18:3/supersensitiveC-reactive protein, LPC 16:0/supersensitive C-reactive protein, PC-016:0/24-alkyl/CRP, PC 16:0/22:6/supersensitive C-reactive protein and CE20:5/supersensitive C-reactive protein (Table 6).

For the purposes of the invention, and particularly for lipid-clinicalconcentration ratios, an Apolipoprotein A-I measurement mayalternatively be an Apolipoprotein A-II measurement.

In a further aspect, the invention relates to a method of choosing anappropriate treatment of CVD and/or one or more of its complications,such as AMI or CVD death, in a subject undergoing statin treatment andnot suffering from type 2 diabetes mellitus, said method comprisingdetermining in a sample from said subject the concentration(s) of one ormore lipid(s), wherein (an) increased or decreased concentration(s) insaid sample, when compared to a control sample, is (are) indicative ofsaid subject being in need of treatment or a change in, orsupplementation of, an already administered treatment, wherein the oneor more lipid(s) whose increase(s) in concentration is (are) compared tothe control is (are) selected from: SM (d18:1/17:0) (d18:1/16:1-OH), Cer18:1/24:0, PC 16:0/22:6, GluCer 18:1/24:1 and CE 18:3 (Tables 4a, 7 and9); and wherein the one or more lipid(s) whose decrease(s) inconcentration is (are) compared to the control is (are) selected from:

GluCer 18:1/18:0, Cer 18:1/16:0, Cer 18:1/24:1 and PC 18:0/18:1 (Table9).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: Cer 18:1/24:0 and GluCer 18:1/24:1 (Tables 4a, 7 and 9).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:Cer 18:1/24:0, PC 16:0/22:6, GluCer 18:1/24:1 and CE 18:3 (Table 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: GluCer 18:1/18:0, Cer18:1/16:0, Cer 18:1/24:1 and PC 18:0/18:1 (Table 9).

In a particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: Cer 18:1/24:0 and GluCer 18:1/24:1 (Table 9).

In an alternative embodiment, the present invention relates to a methodof choosing an appropriate treatment of CVD and/or one or more of itscomplications, such as AMI or CVD death, in a subject undergoing statintreatment and not suffering from type 2 diabetes mellitus, said methodcomprising determining in a sample from said subject one or morelipid-lipid concentration ratio(s), wherein (an) increased or decreasedlipid-lipid concentration ratio(s) in said sample, when compared to acontrol sample, is (are) indicative of said subject being in need oftreatment or a change in, or supplementation of, an already administeredtreatment, wherein the one or more lipid-lipid concentration ratio(s)whose increase(s) is (are) compared to the control is (are) selectedfrom: Cer(d18:1/26:1)/SM (d18:1/24:0) (d18:1/23:1-OH), CE 16:1/SM(d18:1/24:0) (d18:1/23:1-OH), PC 16:0/16:1/PC O-16:0/20:4-alkyl, PC16:0/16:1/PC 18:2/18:2, CE 16:1/CE 20:5, PC 16:0/16:1/PC 18:1/20:4, PC18:0/18:1/PC 18:2/18:2, Cer(d18:1/18:0)/PC O-16:0/20:4-alkyl,Cer(d18:1/20:0)/PC 18:2/18:2, Cer(d18:1/20:0)/PC O-16:0/20:4-alkyl, CE16:1/CE 20:4, Cer(d18:1/24:1)/PC 18:2/18:2, Cer(d18:1/24:1)/PC18:1/20:4, CE 16:1/PC 18:2/18:2, CE 16:1/CE 18:3, Cer(d18:1/18:0)/PCO-16:0/18:2-alkyl, CE 17:1/CE 20:4, PC 16:0/18:1/PC 18:1/20:4, PC16:0/18:1/PC O-16:0/20:4-alkyl, CE 16:1/PC O-16:0/20:4-alkyl, PC18:1/18:1/PC 18:1/20:4, SM (d18:1/16:1) (d18:1/15:2-OH)/SM (d18:1/24:0)(d18:1/23:1-OH), CE 18:1/CE 20:4, Cer(d18:1/18:0)/Cer(d18:1/24:0),Cer(d18:1/24:1)/PC O-16:0/18:2-alkyl, PC 18:0/18:1/PC 18:0/20:3,Cer(d18:1/26:1)/PC 16:0/22:6, CE 16:1/PC 18:1/20:4, Cer(d18:1/26:1)/PC18:0/18:2, CE 16:1/Cer(d18:1/24:0), Cer(d18:1/26:1)/PC 18:1/18:2,Cer(d18:1/26:1)/PC 16:0/18:2, PC 18:1/18:2/PC 18:2/18:2 and PC16:0/18:2/PC 18:2/18:2 (Table 4b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE14:0/CE 16:1, CE 18:3/PC 16:0/18:1, CE 14:0/PC 18:0/18:1,Cer(d18:1/24:0)/Cer(d18:1/26:1), CE 20:4/PC 18:0/18:1,Gb3(d18:1/22:0)/PC 16:0/18:1, CE 18:3/PC 18:0/18:1, CE 20:5/PC16:0/16:1, CE 20:4/Cer(d18:1/26:1), CE 18:3/PC 16:0/16:1, CE18:3/Cer(d18:1/26:1), LPC 16:0/SM (d18:1/17:0) (d18:1/16:1-OH),Gb3(d18:1/22:0)/SM (d18:1/17:0) (d18:1/16:1-OH) and CE 20:5/CE 22:2(Table 4b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from:

Cer(d18:1/26:1)/SM (d18:1/24:0) (d18:1/23:1-OH) and PC 16:0/16:1/PC18:1/20:4 (Table 7); and the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: Gb3(d18:1/22:0)/PC 16:0/18:1, CE 20:4/Cer(d18:1/26:1) andCE 20:5/CE 22:2 (Table 7).

In yet another alternative embodiment the present invention relates to amethod of choosing an appropriate treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject undergoingstatin treatment and not suffering from type 2 diabetes mellitus, saidmethod comprising determining in a sample from said subject one or morelipid-clinical concentration ratio(s), wherein (an) increased ordecreased lipid-clinical concentration ratio(s) in said sample, whencompared to a control sample, is (are) indicative of said subject beingin need of treatment or a change in, or supplementation of, an alreadyadministered treatment, wherein the one or more lipid-clinicalconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: SM (d18:1/17:0) (d18:1/16:1-OH)/HDLcholesterol, CE 22:2/HDL cholesterol, SM (d18:1/17:0)(d18:1/16:1-OH)/apolipoprotein A-I, PC 16:0/16:1/apolipoprotein A-I, CE22:2/apolipoprotein A-I, PC 16:0/16:1/HDL cholesterol,Cer(d18:1/26:1)/HDL cholesterol, Cer(d18:1/26:1)/apolipoprotein A-I, PC18:0/18:1/apolipoprotein A-I, PC 18:0/18:1/total cholesterol, PC16:0/18:1/apolipoprotein A-I, PC 16:0/18:1/apolipoprotein A-I,Cer(d18:1/26:1)/LDL cholesterol, PC 16:0/18:1/total cholesterol, PC18:1/18:1/apolipoprotein A-I, Cer(d18:1/26:1)/total cholesterol, CE15:0/HDL cholesterol, PC 18:1/18:1/HDL cholesterol, CE15:0/apolipoprotein A-I, PC 18:1/18:1/total cholesterol, CE 17:1/HDLcholesterol, CE 17:1/apolipoprotein A-I, Cer(d18:1/26:1)/apolipoproteinB, PC 16:0/18:1/LDL cholesterol, CE 22:6/HDL cholesterol and PC16:0/18:2/LDL cholesterol (Table 4c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: SM(d18:1/24:0) (d18:1/23:1-OH)/triglycerides, CE 19:1 (oxCE682.6)/supersensitive C-reactive protein, PC 17:0/18:2/supersensitiveC-reactive protein, SM (d18:1/17:0) (d18:1/16:1-OH)/supersensitiveC-reactive protein, Gb3 (d18:1/24:1)/supersensitive C-reactive protein,Gb3 (d18:1/22:0)/supersensitive C-reactive protein, SM (d18:1/15:0)(d18:1/14:1-OH)/supersensitive C-reactive protein, SM (d18:1/23:1)(d18:1/22:2-OH)/supersensitive C-reactive protein, CE22:6/supersensitive C-reactive protein, CE 15:0/supersensitiveC-reactive protein, SM (d18:1/16:0) (d18:1/15:1-OH)/supersensitiveC-reactive protein, PC 16:0/18:2/supersensitive C-reactive protein, SM(d18:1/24:1) (d18:1/23:2-OH)/supersensitive C-reactive protein and PC18:2/18:2/supersensitive C-reactive protein (Table 4c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: SM (d18:1/17:0) (d18:1/16:1-OH)/HDL cholesterol, CE22:2/HDL cholesterol and PC 16:0/16:1/apolipoprotein A-I (Table 7);

and the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from:Gb3(d18:1/22:0)/supersensitive C-reactive protein, CE22:6/supersensitive C-reactive protein and PC 18:2/18:2/supersensitiveC-reactive protein (Table 7).

In another aspect, the present invention relates a method of choosing anappropriate treatment of CVD and/or one or more of its complications,such as AMI or CVD death, in a subject undergoing statin treatment whois suffering from type 2 diabetes mellitus, said method comprisingdetermining in a sample from said subject the concentration(s) of one ormore lipid(s), wherein (an) increased or decreased concentration(s) insaid sample, when compared to a control sample, is (are) indicative ofsaid subject being in need of treatment or a change in, orsupplementation of, an already administered treatment, wherein the oneor more lipid(s) whose increase(s) in concentration is (are) compared tothe control is (are) selected from: CE 19:1 (oxCE 682.6), CE 20:0, Cer18:1/16:0, CE 16:0, PC 16:0/18:2, Cer 18:1/20:0, and SM 18:1/24:1(Tables 5a, 8 and 9);

and wherein the one or more lipid(s) whose decrease(s) in concentrationis (are) compared to the control is (are) selected from: PC 18:2/18:2,

CE 22:6, Cer(d18:1/24:0), PC 18:0/22:6, PC P-18:0/20:4, CE 18:1, PCO-16:0/20:4-alkyl, LPC 16:0, DAG 16:0/18:2, CE 20:4, PC 18:1/20:4, PC16:0/16:1, DAG 16:0/18:1, CE 16:1, CE 18:3, CE 20:5, Cer 18:1/18:0, CE20:3, PC 16:0/16:0, Cer 18:1/22:0, PC 18:1/18:2, CE 16:1, CE 18:1 and CE20:4 (Tables 5a, 8 and 9).

In one particular embodiment, the one or more lipid(s) whose increase(s)in concentration is (are) compared to the control is (are) selectedfrom: CE 19:1 (oxCE 682.6), CE 20:0, Cer 18:1/16:0, PC 16:0/18:2, Cer18:1/20:0, and SM 18:1/24:1 (Tables 5a, 8 and 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: PC 18:2/18:2, CE 22:6,Cer(d18:1/24:0), PC 18:0/22:6, PC P-18:0/20:4, CE 18:1, PCO-16:0/20:4-alkyl, LPC 16:0, DAG 16:0/18:2, CE 20:4, PC 18:1/20:4, DAG16:0/18:1, CE 16:1, CE 20:5, Cer 18:1/18:0, PC 16:0/16:0, Cer 18:1/22:0,PC 18:1/18:2, CE 16:1, CE 18:1 and CE 20:4 (Tables 5a, 8 and 9).

In a preferred embodiment, the one or more lipid(s) whose increase(s) inconcentration is (are) compared to the control is (are) selected from:Cer 18:1/16:0, CE 16:0, PC 16:0/18:2, Cer 18:1/20:0 and SM 18:1/24:1(Table 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 20:5, Cer 18:1/18:0,CE 20:3, PC 16:0/16:0, Cer 18:1/22:0, PC 18:1/18:2, CE 16:1, CE 18:1 andCE 20:4 (Table 9).

In a particularly preferred embodiment, the one or more lipid(s) whoseincrease(s) in concentration is (are) compared to the control is (are)selected from: Cer 18:1/16:0, PC 16:0/18:2, Cer 18:1/20:0 and SM18:1/24:1 (Table 9);

and the one or more lipid(s) whose decrease(s) in concentration is (are)compared to the control is (are) selected from: CE 20:5, Cer 18:1/18:0,PC 16:0/16:0, Cer 18:1/22:0, PC 18:1/18:2, CE 16:1, CE 18:1 and CE 20:4(Table 9).

In an alternative embodiment, the present invention relates to a methodof choosing an appropriate treatment of CVD and/or one or more of itscomplications, such as AMI or CVD death, in a subject undergoing statintreatment who is suffering from type 2 diabetes mellitus, said methodcomprising determining in a sample from said subject one or morelipid-lipid concentration ratio(s), wherein (an) increased or decreasedlipid-lipid concentration ratio(s) in said sample, when compared to acontrol sample, is (are) indicative of said subject being in need oftreatment or a change in, or supplementation of, an already administeredtreatment, wherein the one or more lipid-lipid concentration ratio(s)whose increase(s) is (are) compared to the control is (are) selectedfrom: Cer(d18:1/16:0)/DAG 16:0/18:1, Cer(d18:1/16:0)/PC 18:0/20:3,Cer(d18:1/20:0)/PC 18:0/20:3, PC 16:0/16:0/PC 18:1/20:4, CE 15:0/CE18:3, CE 15:0/CE 16:1, PC 17:0/18:2/PC 18:1/20:4, PC 16:0/18:2/PC18:1/20:4 and PC 18:0/18:2/PC 18:1/20:4 (Table 5b);

and wherein the one or more lipid-lipid concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: CE18:1/SM (d18:1/24:1) (d18:1/23:2-OH), PC P-18:0/20:4/SM (d18:1/24:1)(d18:1/23:2-OH), CE 18:1/Cer(d18:1/16:0), CE 16:1/CE 17:1, CE 18:3/PC18:0/18:2, PC 18:0/20:3/SM (d18:1/16:0) (d18:1/15:1-OH), CE18:3/GlcCer(d18:1/16:0), CE 18:3/PC 16:0/18:2, CE 14:0/Gb3(d18:1/16:0),PC 18:1/20:4/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:3/PE 18:0/18:2, PC 18:1/20:4/PCO-18:0/18:2-alkyl, CE 20:4/SM (d18:1/24:1) (d18:1/23:2-OH), CE16:1/Gb3(d18:1/16:0), CE 20:4/PC 17:0/18:2, PC 18:1/20:4/SM (d18:1/24:1)(d18:1/23:2-OH), CE 18:3/SM (d18:1/16:1) (d18:1/15:2-OH), CE 16:1/SM(d18:1/16:0) (d18:1/15:1-OH), CE 18:3/Gb3(d18:1/22:0), CE18:3/LacCer(d18:1/16:0), CE 18:3/SM (d18:1/16:0) (d18:1/15:1-OH), PC18:1/20:4/SM (d18:1/15:0) (d18:1/14:1-OH), CE 18:3/Gb3(d18:1/16:0), CE18:3/SM (d18:1/18:0), CE 16:1/SM (d18:1/24:1) (d18:1/23:2-OH), CE16:1/Cer(d18:1/16:0), CE 20:5/LacCer(d18:1/16:0), CE 18:3/SM(d18:1/24:1) (d18:1/23:2-OH), CE 18:3/SM (d18:1/15:0) (d18:1/14:1-OH),CE 20:5/Gb3(d18:1/16:0) and CE 20:5/SM (d18:1/24:1) (d18:1/23:2-OH)(Table 5b); or

In one particular embodiment, the one or more lipid-lipid concentrationratio(s) whose decrease(s) is (are) compared to the control is (are)selected from: CE 18:1/SM (d18:1/24:1) (d18:1/23:2-OH), PCP-18:0/20:4/SM (d18:1/24:1) (d18:1/23:2-OH), CE 16:1/CE 17:1, CE 18:3/PC18:0/18:2, PC 18:0/20:3/SM (d18:1/16:0) (d18:1/15:1-OH), CE18:3/GlcCer(d18:1/16:0), CE 18:3/PC 16:0/18:2, CE 14:0/Gb3(d18:1/16:0),PC 18:1/20:4/SM (d18:1/16:0) (d18:1/15:1-OH), CE 18:3/SM (d18:1/14:0)(d18:1/13:1-OH), CE 18:3/PE 18:0/18:2, PC 18:1/20:4/PCO-18:0/18:2-alkyl, CE 20:4/SM (d18:1/24:1) (d18:1/23:2-OH), CE16:1/Gb3(d18:1/16:0), CE 20:4/PC 17:0/18:2, PC 18:1/20:4/SM (d18:1/24:1)(d18:1/23:2-OH), CE 18:3/SM (d18:1/16:1) (d18:1/15:2-OH), CE 16:1/SM(d18:1/16:0) (d18:1/15:1-OH), CE 18:3/Gb3(d18:1/22:0), CE18:3/LacCer(d18:1/16:0), CE 18:3/SM (d18:1/16:0) (d18:1/15:1-OH), PC18:1/20:4/SM (d18:1/15:0) (d18:1/14:1-OH), CE 18:3/Gb3(d18:1/16:0), CE18:3/SM (d18:1/18:0), CE 16:1/SM (d18:1/24:1) (d18:1/23:2-OH), CE20:5/LacCer(d18:1/16:0), CE 18:3/SM (d18:1/24:1) (d18:1/23:2-OH), CE18:3/SM (d18:1/15:0) (d18:1/14:1-OH), CE 20:5/Gb3(d18:1/16:0) and CE20:5/SM (d18:1/24:1) (d18:1/23:2-OH) (Table 5b).

In a preferred embodiment, the one or more lipid-lipid concentrationratio(s) whose increase(s) is (are) compared to the control is (are)selected from: Cer(d18:1/16:0)/DAG 16:0/18:1, Cer(d18:1/16:0)/PC18:0/20:3, PC 17:0/18:2/PC 18:1/20:4 and PC 16:0/18:2/PC 18:1/20:4(Table 8).

In yet another alternative embodiment the present invention relates to amethod of choosing an appropriate treatment of CVD and/or one or more ofits complications, such as AMI or CVD death, in a subject undergoingstatin treatment who is suffering from type 2 diabetes mellitus, saidmethod comprising determining in a sample from said subject one or morelipid-clinical concentration ratio(s), wherein (an) increased ordecreased lipid-clinical concentration ratio(s) in said sample, whencompared to a control sample is (are) indicative of said subject beingin need of treatment or a change in, or supplementation of, an alreadyadministered treatment, wherein the one or more lipid-clinicalconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: CE 20:0/apolipoprotein A-I, CE20:0/total cholesterol, SM (d18:1/24:1) (d18:1/23:2-OH)/apolipoproteinA-I, SM (d18:1/24:1) (d18:1/23:2-OH)/HDL cholesterol, SM (d18:1/24:1)(d18:1/23:2-OH)/total cholesterol, Gb3(d18:1/16:0)/HDL cholesterol, SM(d18:1/16:0) (d18:1/15:1-OH)/apolipoprotein A-I, PCO-18:0/18:2-alkyl/apolipoprotein A-I, Gb3(d18:1/16:0)/apolipoproteinA-I, SM (d18:1/24:1) (d18:1/23:2-OH)/triglycerides, SM (d18:1/15:0)(d18:1/14:1-OH)/triglycerides, Gb3(d18:1/16:0)/triglycerides, PC17:0/18:2/triglycerides and Gb3(d18:1/16:0)/total cholesterol (Table5c);

and wherein the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is (are) selected from: PCP-18:0/20:4/apolipoprotein B, CE 20:4/apolipoprotein A-I, CE 14:0/HDLcholesterol, PC 18:1/20:4/HDL cholesterol, CE 20:4/total cholesterol, PC16:0/16:1/apolipoprotein B, PC 18:1/20:4/total cholesterol, CE20:4/apolipoprotein B, PC 18:0/20:3/apolipoprotein B, DAG16:0/18:1/apolipoprotein B, CE 20:3/LDL cholesterol, PC18:1/20:4/apolipoprotein B, PC 18:0/20:3/LDL cholesterol, CE 20:4/LDLcholesterol, CE 20:5/total cholesterol, PC 18:1/20:4/LDL cholesterol, PCP-16:0/18:2/supersensitive C-reactive protein, DAG16:0/18:2/supersensitive C-reactive protein, CE 19:2 (oxCE680.6)/supersensitive C-reactive protein and PCP-18:0/20:4/supersensitive C-reactive protein (Table 5c).

In a preferred embodiment, the one or more lipid-clinical concentrationratio(s) whose increase(s) is (are) compared to the control is: CE20:0/apolipoprotein A-I (Table 8);

and the one or more lipid-clinical concentration ratio(s) whosedecrease(s) is (are) compared to the control is: DAG16:0/18:2/supersensitive C-reactive protein (Table 8).

In connection with all aspects and embodiments of the inventiondescribed and claimed herein, the determination of the lipidconcentration(s), the lipid-lipid concentration ratio(s) or thelipid-clinical concentration ratio(s) is typically performed using anassay.

In one embodiment of the invention, in which the treatment effectivenessis to be evaluated or the treatment is to be chosen as appropriate inaccordance with the methods described and claimed herein, said treatmentis a lipid modifying treatment.

For the purposes of the invention, at least one lipid concentration,lipid-lipid concentration ratio or lipid-clinical concentration ratiofrom Tables 3-8, or combinations thereof, may be determined to assesswhether the patient is at risk to develop one or more of CVDcomplications, such as AMI or CVD death; to evaluate the effectivenessof the treatment of CVD and/or one or more of its complications, such asAMI or CVD death in a subject; or to choose an appropriate treatment ofCVD and/or one or more of its complications, such as AMI or CVD death ina subject. However, it is also possible, and may be advantageous, todetermine at least 2, at least 3, at least 4, at least 5, at least 6, atleast 7, or at least 8 lipid concentrations, lipid-lipid concentrationratios or lipid-clinical concentration ratios from Tables 3-8, orcombinations thereof, in this regard. Where more than one lipidomicmarkers are determined and used for the assessment, it may beadvantageous that a specific lipid concentration, lipid-lipidconcentration ratio, lipid-clinical concentration ratio or combinationthereof, is given greater weight than others in the above-mentionedassessment, evaluation or choice.

In the context of the present invention, CVD is typically characterizedby coronary artery disease, peripheral artery disease, a stroke and/orCVD death. The CVD in the subject whose sample is analyzed in accordancewith the invention may be atherosclerosis-induced. Generally, theinvention embodies methods involving subjects who are at risk ofdeveloping CVD, and have atherosclerosis. Alternatively, the inventionembodies methods involving subjects who are at risk of developing CVD,and do not have atherosclerosis.

In a further embodiment, the methods of the invention may furthercomprise determining the serum or plasma level of total cholesterol,low-density lipoprotein cholesterol (LDL-C), high-density lipoproteincholesterol (HDL-C), Apolipoprotein B (ApoB) and/or Apolipoprotein C-IIIin the subject's sample. In one embodiment of the invention, the subjectdoes not have elevated serum or plasma levels of one or more of totalcholesterol, low-density lipoprotein cholesterol (LDL-C), ApolipoproteinC-III or Apolipoprotein B (ApoB), or a decreased serum level ofHDL-cholesterol (HDL-C).

In accordance with all aspects and embodiments described and claimedherein, both the sample from the subject and the control sample ispreferably a blood sample, more preferably a blood plasma sample, oralso preferably a blood serum sample. It may also be urine or tissue ora fraction of blood, blood plasma, blood serum, urine or tissue e.g., alipoprotein fraction. A blood sample can be prepared and plasma orserum, or fractions thereof, can be separated therefrom with techniqueswell known to the person skilled in the art. Alternatively, both thesample from the subject and the control sample may also be a tissuesample, e.g., artery tissue, such as carotid artery tissue, or arteryplaque material, such as carotid artery plaque material.

Collecting information on a lipidomic marker (i.e., the concentration(s)of (a) lipid(s), lipid-lipid concentration ratio(s), or lipid-clinicalconcentration ratio(s) or combinations thereof, i.e., correspondingprofile(s)) from the sample of a patient and, where appropriate, acorresponding control sample, can be performed with various chemical andhigh-resolution analytical techniques. Suitable analytical techniquesinclude, but are not limited to, mass spectrometry and nuclear resonancespectroscopy. Any high-resolution technique capable of resolvingindividual lipids or lipid classes and providing structural informationof the same can be used to collect the information on the lipidomicmarker in question, e.g., lipid profile from the biological sample. Formethods of the present invention the level of the lipid is determined byusing mass spectrometry, nuclear magnetic resonance spectroscopy,fluorescence spectroscopy or dual polarisation interferometry, a highperformance separation method such as HPLC, UHPLC or UPLC and/or animmunoassay such as an ELISA. According to an alternative or furtherembodiment an analyte in a sample can be detected and/or quantified bycombining the analyte with a binding moiety capable of specificallybinding the analyte. The binding moiety can include, for example, amember of a ligand-receptor pair, i.e., a pair of molecules capable ofhaving a specific binding interaction. The binding moiety can alsoinclude, for example, a member of a specific binding pair, such asantibody-antigen, enzyme-substrate, nucleic acid-based ligands, otherprotein ligands, or other specific binding pairs known in the art. In apreferred embodiment, the lipidomic profile is collected with massspectrometry (MS), wherein the MS instrument may be coupled to directinfusion methods and high performance separation methods such as HPLC,UPLC or UHPLC. The amount of the individual lipids or lipid classes inthe collected lipidomic profile is used when comparing the collectedlipid profile to a control.

Collecting the information on the lipidomic marker with massspectrometry (MS) is one of the preferred embodiments of the currentinvention. The MS instrument can be coupled to a direct sample infusionmethod, such as a robotic nanoflow ion source device, or to a highperformance separation method such as high performance liquidchromatography (HPLC), ultra performance liquid chromatography (UPLC) orultra high performance liquid chromatography (UHPLC).

The methods of the present invention may be used for determining a riskof said patient to develop CVD complications, particularly severe CVDcomplications such as death and myocardial infarction (MI), includingacute myocardial infarction (AMI).

In one embodiment of the invention, a method for treating or preventingCVD complications, such as AMI or CVD death, in a subject in needthereof is provided. The method comprises administering atherapeutically effective dose of a drug capable of modulating one ormore of the lipid concentration(s), lipid-lipid concentration ratio(s)or lipid-clinical concentration ratio(s) described in Tables 3-8,wherein the dose is such that said one or more lipid concentration(s),lipid-lipid concentration ratio(s) or lipid-clinical concentrationratio(s) in a sample of said subject does not significantly differ whencompared to (a) corresponding lipid concentration(s), (a) correspondinglipid-lipid concentration ratio(s) or (a) corresponding lipid-clinicalconcentration ratio(s) in a control, e.g., a control sample. In apreferred embodiment, the drug is a statin or another HMG-CoA reductaseinhibitor. Particularly preferred statins in this regard areatorvastatin, cerivastatin, fluvastatin, fluvastatin XL, lovastatin,mevastatin, pitavastatin, pravastatin, rosuvastatin or simvastatin. Inanother preferred embodiment, the drug is niacin (nicotinic acid); acholesterol absorption inhibitor, such as ezetimibe or SCH-48461; acholesteryl ester transfer protein (CETP) inhibitor, such astorcetrapib, anacetrapib or JTT-705; a bile acids sequestrant, such ascolesevelam, cholestyramine or colestipol; or a fibrate, such asfenofibrate, gemfibrozil, clofibrate, or bezafibrate. Alternatively, itmay also be a phytosterol or a PCSK9 inhibitor.

Also part of the present invention is a lipid as described herein, e.g.a lipid from any of Tables 3, 4, 5, 6, 7 or 8, for use in preventing ortreating a subject at risk to develop CVD complications such as AMI orCVD death. The said lipid may be taken as a dietary supplement or amedicament. A corresponding method of treatment is likewise encompassed.Likewise, the invention also encompasses a modulator for use formodulating a lipid concentration, lipid-lipid concentration ratio orlipid-clinical concentration ratio as described and/or claimed herein,e.g., in Tables 3-8, in a subject at risk to develop CVD and/or one ormore of its complications such as AMI or CVD death. A correspondingmethod of treatment is likewise encompassed. In a further embodiment,the said modulator is a small molecule, an antisense RNA, a smallinterfering RNA (siRNA) or a natural or modified lipid.

Alternatively, the modulator affects the activity, functionality orconcentration of an enzyme, wherein said enzyme catalyzes a reactionthat produces or degrades any one of the lipids in Tables 3-9.Similarly, the present invention relates to a preventing or treating asubject at risk to develop CVD complications such as AMI or CVD death,using, or administering a modulator, wherein the modulator affects theactivity, functionality or concentration of an enzyme, wherein saidenzyme catalyzes a reaction that produces or degrades any one of thelipids in Tables 3-9.

In one embodiment of the present invention, an antibody against any oneof the lipids in Tables 3-9 is used for predicting one or more CVDcomplications such as AMI or CVD death. In another embodiment of theinvention, the antibody may be used for preventing or treating one ormore of the above complications in a subject.

Any of the methods, drugs, lipids, modulators or antibodies of thepresent invention may be used for a subject which is at risk to developor has suffered from one or more CVD complications such as acutemyocardial infarction and/or is at risk of cardiovascular death. For thepurposes of the invention, CVD complication(s) includes severe CVDcomplication(s), particularly death.

Also encompassed by the present invention is a kit for predicting CVDand/or one or more of it complications, or for performing the methods oruses described and/or claimed herein, wherein the kit comprises reagentsand reference compounds. The reference compounds may be one or more ofthe following, but are not limited to: (a) (a) lipid standard(s) chosenfrom the lipids in Tables 3 to 9, (b) one or more control markers (forexample, a lipid or lipids, preferably a lipid corresponding to any ofthe lipidomic markers described and/or claimed herein, or (an)otherlipid(s), e.g., total PC, or another molecule, e.g., a protein; c)positive and/or negative controls; d) internal and/or externalstandards; e) calibration line controls; (f) an antibody or otherbinding moiety capable of binding any one of the lipids in Tables 3 to9. The reagents are solution(s), solvent(s), and/or buffer(s) useful forperforming said methods or uses.

In one embodiment of the invention, a kit is provided for predicting CVDand/or one or more of its complications, or for performing the methodsof the invention, wherein the kit comprises (a) (a) lipid standard(s)chosen from the lipids in Tables 3 to 9, and optionally one or morefurther reference compound(s) selected from: (b) one or more controlmarkers (for example, a lipid or lipids, preferably a lipidcorresponding to any of the lipidomic markers described and/or claimedherein, or another lipid(s), e.g., total PC, or another molecule, e.g.,a protein); c) positive and/or negative controls; d) internal and/orexternal standards, which may or may not be chemically modified, taggedor non-endogenously occurring molecules in human; e) calibration linecontrols; and (f) an agent, optionally an antibody, capable of bindingany one of the lipids in Tables 3 to 9, and (g) (a) reagent(s) forperforming said methods or uses.

Preferred kits according to the invention comprise, for example, thefollowing combinations of the above listed constituents: (a) and (b),and optionally (g); (a) and (c), and optionally (g); (a) and (d), andoptionally (g); (a) and (e), and optionally (g); (a) and (f), andoptionally (g); (a), (b) and (c), and optionally (g); (a), (c) and (d),and optionally (g); (a), (d) and (e), and optionally (g); or (a), (e)and (f), and optionally (g).

In one preferred embodiment, the one or more control marker(s) of theclaimed kit is/are (a) molecule(s) that is/are regularly measured in aclinical setting. For example, preferred are embodiments wherein the oneor more said control marker(s) is CK.

Preferably, the kit is used for predicting CVD complications, whereinthe lipid concentration(s), lipid ratio(s) or (a) lipid combination(s)thereof in a sample from a subject is (are) determined by using massspectrometry. The sample may be subjected to purification and/or othersample pre-preparation step(s) before mass spectrometry analysis. Thepurification step may be, but is not limited to chromatography, forexample, high performance liquid chromatography (HPLC), ultraperformance liquid chromatography (UPLC) and/or ultra high performanceliquid chromatography (UHPLC). The sample pre-preparation step may be,but is not limited to solid-phase extraction (SPE), derivatization,liquid-liquid extraction and/or lipoprotein fractionation. The said massspectrometry determination may be done by tandem mass spectrometry.

As mentioned above, for the purposes of the present invention, a controlsample may be obtained from (a) CAD patient(s) or a group of CADpatients that has/have remained free of any major CVD complicationse.g., by mixing a variety of samples from said population. If a group ofCAD patients is used then several lipid profiles from a population arecombined and the lipidomic marker is created from this combination. Thelevels or amounts of the individual lipids or the lipid-lipidconcentration ratios or lipid-clinical concentration ratios in thesample from a subject are compared to the levels or amounts of thelipids or lipid ratios in the control for determining the risk of one ormore of CVD complications, such as AMI or CVD death, in said subject.

In one embodiment, the control is a sample from (a) CAD patient(s)wherein the control sample is from (a) CAD patient(s) or a group of CADpatients with no history of major CVD events and who is/are undergoingstatin treatment. The control CAD patient(s) undergoing statin treatmentmay or may not have a type 2 diabetes mellitus. It may also be a samplethat represents a combination of samples from a CAD patient populationundergoing statin treatment with no history of major CVD events.Alternatively, the control may be a set of data concerning a lipidomicmarker in accordance with the present invention, e.g., information onthe concentration of (a) lipid(s), lipid-lipid concentration ratio(s),or lipid-clinical concentration ratio(s) in accordance with the presentinvention in a sample when taken from (a) CAD patient(s) undergoingstatin treatment with no history of major CVD events, or in acombination of samples taken from a CAD patient population undergoingstatin treatment with no history of major CVD events. Said information,and thus the corresponding set of data, may have been previouslydetermined, calculated or extrapolated, or may have yet to bedetermined, calculated or extrapolated, or may also be taken from theliterature.

Alternatively, the control is a sample from (a) CAD patient(s) whereinthe control sample is from (a) CAD patient(s) or a group of CAD patientswith no history of major CVD events and who is/are not undergoing statintreatment. The control CAD patient(s) not undergoing statin treatmentmay or may not have a type 2 diabetes mellitus. It may also be a samplethat represents a combination of samples from a CAD patient populationnot undergoing statin treatment with no history of major CVD events.Alternatively, the control may be a set of data concerning a lipidomicmarker in accordance with the present invention, e.g., information onthe concentration of (a) lipid(s), lipid-lipid concentration ratio(s),or lipid-clinical concentration ratio(s) in accordance with the presentinvention in a sample when taken from (a) CAD patient(s) not undergoingstatin treatment with no history of major CVD events, or in acombination of samples taken from a CAD patient population notundergoing statin treatment with no history of major CVD events. Saidinformation, and thus the corresponding set of data, may have beenpreviously determined, calculated or extrapolated, or may have yet to bedetermined, calculated or extrapolated, or may also be taken from theliterature.

Preferably, the control sample is blood, plasma, serum, urine or tissue,or a lipoprotein fraction thereof.

In another aspect, the present invention relates to a statin or a lipidlowering drug for use in the treatment of a subject at risk to developone or more CVD complications, such as acute myocardial infarction (AMI)and/or CVD death, wherein said subject would be identified as being atrisk to develop one or more CVD complications when applying any of themethods, drugs, lipids, modulators, kits or uses described and/orclaimed herein. Similarly, the present invention relates to a method oftreating a subject at risk to develop one or more CVD complications witha statin or a lipid lowering drug, wherein said subject would beidentified as being at risk to develop one or more CVD complicationswhen applying any of the methods, drugs, lipids, modulators, kits oruses described and/or claimed herein.

In a further embodiment, the present invention relates to a statin or alipid lowering drug for use in the treatment of a subject at risk todevelop one or more CVD complications, such as AMI and/or CVD death,wherein said subject actually has been identified as being at risk todevelop one or more CVD complications by any of the methods, drugs,lipids, modulators, kits or uses described and/or claimed herein.Similarly, the present invention relates to a method of treating asubject at risk to develop one or more CVD complications with a statinor a lipid lowering drug, wherein said subject actually has beenidentified as being at risk to develop one or more CVD complications byany of the methods, drugs, lipids, modulators, kits or uses describedand/or claimed herein.

In yet another aspect, the present invention relates to a statin or alipid lowering drug for use in the treatment of a subject at risk todevelop one or more CVD complications, such as AMI and/or CVD death,wherein said subject would be identified as not being at risk to developor as not suffering from one or more CVD complications when applying anyof the methods, drugs, lipids, modulators, kits or uses described and/orclaimed herein. Similarly, the present invention relates to a method oftreating a subject at risk to develop one or more CVD complications witha statin or a lipid lowering drug, wherein said subject would beidentified as not being at risk to develop one or more CVD complicationswhen applying any of the methods, drugs, lipids, modulators, kits oruses described and/or claimed herein.

In a further embodiment, the present invention relates to a statin or alipid lowering drug for use in the treatment of a subject at risk todevelop one or more CVD complications, such as AMI and/or CVD death,wherein said subject actually has been identified as not being at riskto develop or as not suffering from statin-induced muscle toxicity byany of the methods, drugs, lipids, modulators, kits or uses describedand/or claimed herein. Similarly, the present invention relates to amethod of treating a subject at risk to develop one or more CVDcomplications with a statin or a lipid lowering drug, wherein saidsubject actually has been identified as not being at risk to develop oneor more CVD complications by any of the methods, drugs, lipids,modulators, kits or uses described and/or claimed herein.

In the present invention herein, lipid biomarker concentrations havebeen measured and quantified in patients with documented CAD who did notshow fatal outcomes during the follow-up period (3 years) and inhigh-risk CAD patients who died due to cardiovascular events during thefollow-up period. This invention thus enables accurate usage of thelipid-based biomarkers to identify high risk CVD/CAD patients. Anotherlayer of accuracy was reached through a careful patient selection sinceit is important to control for factors which may affect the lipidconcentration read-outs. We used specific targeted platforms on asingular technology set-up to analyze lipid species in particular.

The technology and the way it was applied in the context of theinventive teaching presented herein is set apart from similar efforts inthe field inter alia due to the following criteria. In samplepreparation, samples are strictly controlled and treated identically toavoid potential artifacts that could arise from improper handling. Inconnection with the present invention, samples were carefully thawedslowly on ice and directly thereafter subjected to a custom-madeautomated lipid extraction which possesses currently the highestprecision in liquid handling, therefore minimizing potential errors.Furthermore, sample freeze-thaw cycles were strictly controlled sincethis can dramatically affect the lipid stabilities. The automated lipidextraction is based on the method by Folch and colleagues (Folch J, etal: A simple method for the isolation and purification of total lipidsfrom animal tissues. J Biol Chem 1957, 226(1):497-509) which useschloroform and methanol. This method is preferred when a wide range,from polar to non-polar, of lipid classes are to be extracted withoptimal recoveries thus preventing the loss of lipid species. Lipidclass specific non-endogenous lipids, when applicable, were used asinternal standards to gain highest precision in identification(minimizing false positives) and quantification of monitored molecularlipid species. In this way absolute or semi-absolute amounts ofendogenous molecular lipids were determined with the highest precisionthat can be achieved with today's technologies. The endogenous lipidsand respective standards were monitored at the molecular lipid level. Inthis way, not only false positive identifications were minimized, butmolecular lipids could be precisely determined and quantified. Analysisquality was strictly controlled using a novel quality control system.This was mainly controlled by multiple internal standards (IS), externalstandards (ES), IS/ES ratios, and instrument control samples. Bystringently controlling these components, technical and biologicaloutliers were readily identified and rejected from further analysis. Toobtain best precision in sensitivity, selectivity and quantification foreach molecular lipid different targeted platforms were used. Some lipidsare best analyzed using high performance liquid chromatography (HPLC),ultra performance liquid chromatography (UPLC) or ultra high performanceliquid chromatography (UHPLC) combined with mass spectrometry basedmultiple reaction monitoring (MRM) whereas others are best analyzed bydirect infusion in combination with mass spectrometry-based precursorion scanning and neutral loss scanning techniques.

DETAILED DESCRIPTION OF THE INVENTION

Definitions:

Coronary vascular disease/cardiovascular disease (CVD) has its generalmeaning in the art and is used to classify numerous conditions thataffect the heart, heart valves, blood, and vasculature of the body,including CAD. In the present invention the terms CVD and CAD may beused interchangeably. For the purposes of the invention, CVD/CADpatients, in one embodiment, exclude patients with Acute CoronarySyndrome (ACS). In an alternative embodiment, ACS is included inCVD/CAD. Cardiovascular diseases in accordance with the presentinvention include endothelial dysfunction, coronary artery disease,angina pectoris, myocardial infarction, atherosclerosis, congestiveheart failure, hypertension, cerebrovascular disease, stroke, transientischemic attacks, deep vein thrombosis, peripheral artery disease,cardiomyopathy, arrhythmias, aortic stenosis, and aneurysm. Suchdiseases frequently involve atherosclerosis. In a preferred embodimentof the invention, the cardiovascular disease is a cardiovascular diseaseassociated with atherosclerosis.

CAD is coronary artery disease, AMI is acute myocardial infarction, ACSis acute coronary syndrome, CAC is coronary artery calcification, RCT isreverse cholesterol transport, LDL is low density lipoprotein, HDL ishigh density lipoprotein, LDL-C is low density lipoprotein cholesterol,HDL-C is high density lipoprotein cholesterol, ApoA is Apolipoprotein A,ApoB is Apolipoprotein B, ApoC is apolipoprotein C, MS is massspectrometry, HPLC is high performance liquid chromatography, UHPLC isultra high pressure liquid chromatography and UPLC is ultra performanceliquid chromatography.

As used herein, “a subject” includes all mammals, including withoutlimitation humans, but also non-human primates, dogs, cats, horses,sheep, goats, cows, rabbits, pigs and rodents. A particularly preferred“subject” is a human.

A “sample” is defined as any biological sample obtained from a subjector a group or population of subjects. For the purposes of the presentinvention, the biological sample may be whole blood, blood serum, bloodplasma or a fraction of blood; e.g., a lipoprotein fraction. It may alsobe a tissue sample. However, a preferred embodiment is wherein thebiological sample is plasma or serum. Taking a blood sample of a patientis part of normal clinical practice. The blood sample can be taken inconnection with e.g. measuring the cholesterol levels in the patients.The collected blood sample can be prepared and serum or plasma can beseparated with techniques well known to a person skilled in the art.Vena blood samples can be collected from patients using a needle and aBD Vacutainer® Plastic Tubes or Vacutainer® Plus Plastic Tubes (BDVacutainer® SST™ Tubes contain spray-coated silica and a polymer gel forserum separation). Serum can be separated by centrifugation at 1300 RCFfor 10 min at room temperature and stored in small plastic tubes at −80°C. The lipoprotein fractions may be separated by precipitation,ultracentrifugation, by chromatography or by gel filtration with methodswell known in the art.

For the purposes of the present invention, lipids from the Lipidomicanalysis were named according to the following nomenclature: CE ischolesteryl ester, Cer is ceramide, DAG is diacylglycerol, PC 0 isether-linked PC, Gb3 is globotriaosylceramide, GlcCer is galactosyl- andglucosylceramides, LacCer is lactosylceramides, LPC islysophosphatidylcholine, PC is Phosphatidylcholine, PE isPhosphatidylethanolamine, PI is Phosphatidylinositol and SM isSphingomyelin.

The nomenclature X:Y indicates, X number of total carbon atoms in thefatty acid(s) portions of the molecule, and Y the total number of doublebonds in the fatty acid portion(s) of the molecule.

The nomenclature A/B indicates, for a molecule of DAG and PC, A and Btypes of fatty acid moieties attached to the glycerol backbone of themolecule.

The nomenclature (dC/A) indicates, for a molecule of Cer, Gb3, GlcCer,LacCer and SM, C the type of long-chain base with an amide-linked, A,fatty acid moiety.

According to the invention, “undergoing statin treatment” or “on statintreatment” means that the subject is being treated with one or morestatins and/or any other HMG-CoA reductase inhibitor.

The wording “compared to a control sample” as used herein will beunderstood to include embodiments where control samples are actuallyanalyzed in respect of a lipidomic marker of interest, i.e., in respectof the concentration of one or more of the lipid(s), the lipid-lipidconcentration ratios, or the lipid-clinical concentration ratios orcombinations thereof as specifically described and/or claimed herein inconnection with the various aspects and embodiments of the presentinvention. It will be appreciated, however, that the above wording alsoincludes embodiments where the corresponding information on saidlipidomic marker in said control sample is merely taken from theliterature, or has been previously determined, calculated orextrapolated, or is yet to be determined, calculated or extrapolated.

As used herein, the term “antibody” includes monoclonal and polyclonalantibodies, whole antibodies, antibody fragments, and antibodysub-fragments that exhibit specific binding to a said lipid. Thus,suitable “antibodies” can be whole immunoglobulins of any class, e.g.,IgG, IgM, IgA, IgD, IgE, chimeric antibodies or hybrid antibodies withdual or multiple antigen or epitope specificities, or fragments, e.g.,F(ab′)₂, Fab′, Fab and the like, including hybrid fragments, andadditionally includes any immunoglobulin or any natural, synthetic orgenetically engineered protein that acts like an antibody by binding toa specific antigen to form a complex. The term “antibody” encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab fragments, F(ab′)₂, an Fd fragment, an Fv fragment, a dAb (single(variable) domain antibody), or a nanobody) as well as completeantibodies. For example, Fab molecules can be expressed and assembled ina genetically transformed host like E. coli. A lambda vector system isavailable thus to express a population of Fab's with a potentialdiversity equal to or exceeding that of the predecessor antibody. SeeHuse W D, et al., Science 1989, 246:1275-81. Such Fab's are included inthe definition of “antibody.” The ability of a given molecule, includingan antibody fragment or sub-fragment, to act like an antibody andspecifically bind to a specific antigen can be determined by bindingassays known in the art, for example, using the antigen of interest asthe binding partner.

Antibodies against lipids in accordance with the present invention maybe prepared by methods well known to those skilled in the art. Forexample, mice may be immunized with a lipid with adjuvant. Splenocytesare harvested as a pool from the mice that were administered 3immunizations at 2-week intervals with test bleeds performed onalternate weeks for serum antibody titers. Splenocytes are prepared as 3aliquots that are either used immediately in fusion experiments orstored in liquid nitrogen for use in future fusions.

Fusion experiments are then performed according to the procedure ofStewart & Fuller, J. Immunol. Methods 1989, 123:45-53. Supernatants fromwells with growing hybrids are screened by enzyme-linked immunosorbentassay (ELISA) for monoclonal antibody (MAb) secretors on 96-well ELISAplates coated with the said lipid. ELISA positive cultures are cloned bylimiting dilutions, typically resulting in hybridomas established fromsingle colonies after 2 serial cloning experiments.

EXAMPLES Example 1 Materials and Methods

This study is a sub-cohort of the LURIC study that is a large scaleprospective study on cardiovascular epidemiology. LURIC databasecontains clinical information over 3000 patients including baselinecoronary angiography, clinically used biomarker data and also e.g. CVDmortality data for the follow-up period (3 years). In this biomarkerstudy the inventors compared CAD cases undergoing statin treatment(n=135) that died during the follow-up due to CVD with patients (n=94)having a stable CAD also undergoing statin treatment. Subjects with asignificant atherosclerosis level in the angiogram but no CVD relateddeath during the follow-up were used as controls, while the case grouphad similarly a significant atherosclerosis based on the angiography atbaseline and in addition they died during the follow-up due to acutecardiovascular events. A statistical analysis was performed separatelyfor cases (n=70) and controls (n=65) without diabetes (n=70) and forcases (n=65) and controls (n=29) having diabetes. The clinicalcharacteristics for these two groups are described in Tables 1 and 2.

TABLE 1 Background characteristics of subjects undergoing statintreatment and not having type 2 diabetes mellitus Study group: CADpatients on statin treatment and not having DM2 Controls Cases Variable(n = 65) (n = 70) DM2 patients 0 0 Hypertensive patients 34 (52.3%) 38(54.3%) Smokers (active or quit less than 3 20 (30.8%) 27 (38.6%) yearsbefore sampling) Statin users 65 (100%)  70 (100%)  Age 64.0 65.6apolipoprotein A-I 130.0 121.9 apolipoprotein B 106.5 102.5 Bmi 27.127.3 HDL cholesterol 39.0 36.3 LDL cholesterol 116.0 108.2lipoprotein(a) 31.8 32.0 supersensitive C-reactive protein 3.4 10.3total cholesterol 195.2 184.6 triglycerides 179.9 166.0

TABLE 2 Background characteristics of subjects undergoing statintreatment and having type 2 diabetes mellitus Study group: CAD patientson statin treatment and having DM2 Controls Cases Variable (n = 29) (n =65) DM2 patients 29 (100%) 65 (100%) Hypertensive patients 18 (62.1%) 44(67.7%) Smokers (active or quit less than 3 8 (27.6%) 13 (20%) yearsbefore sampling) Statin users 29 (100%) 65 (100%) Age 63.2 67.5apolipoprotein A-I 132.6 117.7 apolipoprotein B 103.5 99.0 Bmi 28.1 28.2HDL cholesterol 38.1 34.4 LDL cholesterol 102.3 102.6 lipoprotein(a)35.3 31.3 supersensitive C-reactive protein 2.8 19.2 total cholesterol190.4 172.2 triglycerides 230.0 181.7 Definition of Cases: All cases hadsignificant artery disease (>=20% stenosis) in coronary angiogram andthey all died due to CVD during the follow-up. Thus, these CAD patientshave an elevated risk for CVD outcomes. Majority (75%) of cases, werenon-ACS patients. Definition of Controls: All controls had significantartery disease (>=20% stenosis) in coronary angiogram, but they did notdie due to CVD during the follow-up. The control subjects did not haveany history of MI or stroke before the baseline evaluation. Thus, thesepatients can be considered to be CAD patients with a low risk for CVDoutcomes.

Example 2 Analytical Methods

Mass Spectrometry Driven Lipidomics

Direct infusion coupled to tandem mass spectrometry, i.e. shotgunlipidomics, and two liquid chromatography tandem mass spectrometry(LC-MS/MS) approaches, i.e. ceramide and cerebroside lipidomics wereused to identify lipid biomarkers for coronary artery disease (CVD) riskby analyzing molecular lipid species in human serum, plasma, and carotidartery plaques. The applied methods were optimized especially forquantification of molecular cholesteryl esters (CE),phosphatidylcholines (PC), lysophosphatidylcholines (LPC) and otherlysophospholipids (LPL), ether-linked phosphatidylcholines (PC O) andother ether-linked phospholipids (PL O), phosphatidylserines (PS),phosphatidylethanolamines (PE), phosphatidylglycerols (PG),phosphatidylinositols (PI), phosphatidic acids (PA), diacylglycerols(DAG), ceramides (Cer), glucosylceramides (GlcCer),Globotriaosylceramide (Gb3) and lactosylceramides (LacCer).

The following materials were used according to the methods. HPLC orLC-MS grade of chloroform, methanol, water, acetonitrile, formic acid,methanol, isopropanol, ammonium acetate, acetic acid, potassium chlorideand butylated hydroxytoluene (BHT) were purchased from Sigma-Aldrich(St. Louis, Mo., USA).

HPLC column (Acquity BEH C18, 2.1×50 mm id. 1.7 μm) was purchased fromWaters (Milford, Mass., USA). HPLC pre-column (Widepore C18 4×2 0 mm)was purchased from Phenomenex (Torrance, Calif., USA). All labware usedfor the extraction were resistant to chloroform. Aerosol resistantfilter tips (Molecular BioProducts) and Eppendorf 2 ml safe-lock tubes,96-well twin.tec PCR plates, and Pierce-it-lite thermo-sealing foilswere purchased from VWR International (West Chester, Pa., USA). CO-REFilter Tips and 96-well 2 ml Whatman Uniplates were purchased fromHamilton Robotics (Bonaduz, Switzerland). Synthetic lipid standards werepurchased from Avanti Polar Lipids (Alabaster, Ala., USA) and fromMatreya (Pleasant Gap, Pa., USA).

Lipids were extracted in chloroform:methanol according to the followingprotocols. Samples were spiked with known amounts of non-endogenoussynthetic internal standards for data normalization and endogenous lipidquantification. Post-extract spiked non-endogenous synthetic externalstandards were used for quality controlling. Stock solutions ofstandards were prepared by dissolving appropriately weighed amounts ofeach standard in chloroform:methanol (2:1, V:V) to achieve a finalconcentration of 500 μM. An internal standard mixture containing each ofthe standard stock was created and used in lipid extraction.

Samples and quality control samples for each extraction batch werethawed on ice. The carotid artery plaque samples were weighed on ice byusing a cryo-box and homogenized in ice-cold 70% methanol in water. TheMixer Mill 301 Teflon® adapters were kept at −20° C. Homogenization wasperformed at 15-25 Hz for 2-15 minutes with Mixer Mill 301 (Retch GmbH,Germany).

Lipid extraction of human samples was carried out in automated fashionusing a Hamilton MICROLAB STAR system (Hamilton Robotics, Switzerland).Well-mixed samples were aliquoted into a 96-well 2 ml Whatman Uniplatecontaining ice-cold methanol and 0.1% BHT. 5 μl of serum and plasma and30 μl of carotid artery plaques were used for shotgun- and ceramide andcerebroside lipidomics and 100 μl of serum and plasma and 200 μl ofcarotid artery plaques was used for ganglioside lipidomics. The sampleswere mixed thoroughly after each step in the extraction protocol. Theextraction proceeded at room temperature by adding an appropriate volumeof internal standard mixture and chloroform, and methanol and water inthe case of ganglioside lipidomics. In shotgun and ceramide andcerebroside lipidomics, the organic phase separation was facilitated byadding 20 mM acetic acid and centrifuging the plate for 5 min at 500×g.The organic phase was transferred into a new 96-well 2 ml WhatmanUniplate. The remaining water-containing phase was washed by addingappropriate volume of chloroform followed by centrifugation. The twoorganic phases were pooled and evaporated under N₂ until dryness. Thelipid extracts were then re-dissolved in chloroform:methanol (1:2, v:v)including the addition of the synthetic external standard.

In shotgun lipidomics, lipid extracts were analyzed on a hybrid triplequadrupole/linear ion trap mass spectrometer (QTRAP 5500, AB Sciex)equipped with a robotic nanoflow ion source (NanoMate HD, AdvionBiosciences). The instruments were operated in positive and negative ionmodes. In positive ion the spray voltage was set to 1.0 to 1.4 kV and innegative ion mode to −1.0 to −1.4 kV. A gas pressure of 0.3-0.8 psi wasused and the interface heater was set at 60° C. The collision energy(CE) and declustering potential (DP) was optimized for each lipid classusing synthetic standards. The mass spectrometer was operated in unitresolution mode using a scan speed of 200 Da/s. Molecular lipids wereanalyzed in both positive and negative ion modes using multipleprecursor ion scanning (MPIS) and neutral loss scanning (NLS) asdescribed by Ståhlman and colleagues (Ståhlman M, et al. High-throughputshotgun lipidomics by quadrupole time-of-flight mass spectrometry. JChromatogr B Analyt Technol Biomed Life Sci 2009).

In ceramide and cerebroside lipidomics, the high performance liquidchromatography (HPLC) analyses were conducted in the following way.Chromatographic apparatus consisted of a CTC HTC PAL autosampler (CTCAnalytics AG, Switzerland), a Rheos Allegro UPLC pump (Flux InstrumentsAG, Switzerland), an external column heater set to 60° C. for ceramideand cerebroside lipidomics and 45° C. for ganglioside lipidomics, andthe Acquity BEH C18 column with an in-line pre-column. The extractedsamples, 10 μl of each, were injected into the pre-column followed bythe analytical column and delivered to the mass spectrometer at a flowrate of 500 μl/min. In ceramide and cerebroside lipidomics, A gradientwas used for lipid analyte separation with solvent A comprising 10 mMammonium acetate in HPLC grade water containing 0.1% formic acid andsolvent B of 10 mM ammonium acetate in acetonitrile:isopropanol (4:3,V:V) containing 0.1% formic acid. The gradient was constructed in thefollowing way: 0 min-65% B; 2 min-65% B; 2.5 min-75% B; 17.5 min-100% B;22.5 min-100% B; 22.6 min-65% B; 25 min-65% B.

The lipid extracts were analyzed by HPLC-MS/MS. The MS analysis wasperformed on a hybrid triple quadrupole/linear ion trap massspectrometer equipped with the Turbo V™ Ion Source (4000 QTRAP, ABSciex). The instrument was operating in positive and negative ion modes.The ion source voltage was set to 5500V for ceramide and cerebrosidelipidomics and source temperature at 400° C. The collision energy (CE)and declustering potential (DP) was optimized for each lipid class usingsynthetic standards. A 20 sec dwell time was applied for each scan.Multiple reaction monitoring (MRM) scan mode was applied and based onthe description by Sullards and colleagues (Sullards M C, et al:Structure-specific, quantitative methods for analysis of sphingolipidsby liquid chromatography-tandem mass spectrometry: “inside-out”sphingolipidomics. Methods Enzymol 2007).

The data processing was done in the following way. Initially theretention time (in LC mode) and identification of each peak was doneusing endogenous standards and by Information Dependent Acquisition(IDA) experiments where applicable. The raw data were processedaccording to peak detected and retention time (in LC mode) in automatedfashion. A stringent cutoff was applied for separating background noisefrom actual lipid peaks. Each sample was controlled and only acceptedwhen fulfilling the stringent acceptance criteria. Peak area counts(cps) of detected peaks were converted into a list of correspondinglipid names. Lipids were normalized to their respective internalstandard and sample volume or tissue weight to retrieve theirconcentrations.

Several quality controls were used in the lipidomic analyses. Acalibration line using synthetic or isolated standards was obtainedprior to sample analysis. Synthetic standards were chosen based onapplication and had similar properties to the endogenous lipids oranalyte(s) of interest. The calibration line consisted of a minimum offive standards points covering the expected quantification range. Asample extracted without standard and standards extracted with nomatrix, were included with the calibration line.

The calibration line was used to determine the dynamic quantificationrange for each lipid class monitored, e.g., the linear quantificationlimits. As the internal standards used behave in the same way asendogenous lipids they were used for quantifying endogenous lipidspecies. The calibration lines were based on the same internal standardsthat were used for quantification of the endogenous lipids.

In each sample extracted for lipids, the ratio of synthetic internalstandards (IS) to corresponding post-extract spiked external standard(ES) was determined. The peak area (cps) ratio of internal to externalstandard (IS/ES) was used for calculating the Coefficient of Variation(CV) across all samples. The IS/ES ratio enabled the calculation oflipid extraction recovery.

Instrument control (IC) was included at the start, middle and end ofeach run. IC sample analyzed was an extracted reference plasma sampleand a set of standards to monitor the instrument's performance, i.e.,the infra- and inter-assay variation.

For each platform, a stringent cutoff was applied for separatingbackground noise from actual lipid peaks. Each sample was controlled andonly accepted when fulfilling the stringent acceptance criteria. Massesand counts of detected peaks were converted into a list of correspondinglipid names. Lipids were normalized to their respective internalstandard and sample volume to retrieve their concentrations.

Statistical Analyses

Percentage changes in lipid concentrations between control and casegroups were calculated as follows:100*(AVG[C] in case group−AVG[C] in control group)/AVG[C] in controlgroupStatistical significance was assigned based on standard t-test p-values.

In addition, ROC curves were used for finding lipid molecules andconcentration cutoffs that separate the best cases from controls.Selectivity is calculated as a number of correctly identified casesdivided by the total number of cases. Specificity is calculated as anumber of correctly identified controls divided by the total number ofcontrols. Selectivity and specificity was calculated for each lipidconcentration, lipid to lipid ratio and ratio of lipid to clinicalconcentrations.

Example 3 Ethics

The LURIC study was approved by the ethics review committee at the“Landesärztekammer Rheinland-Pfalz” (Mainz, Germany). Written informedconsent was obtained from each of the participants.

Results

In this LURIC study sub-cohort, the traditional biomarkers includingLDL-cholesterol and HDL-cholesterol concentrations were practicallyidentical in both groups and therefore were not predictive ofCVD-related for predicting severe CVD/CAD-associated complications,including AMI, stroke and CVD death in this study.

Multiple lipidomic markers appeared as significant predictors of severeCVD/CAD-associated complications (Tables 3-9). A total of 162 molecularlipids were quantified. The significant predictors were selected basedon the top fifty candidates from each category, when available. Thebiomarker candidates were selected according to the following criteria:t-test p-value≦0.05 or sensitivity≧60% and specificity≧60%. Fromtraditional clinical chemistry only apolipoprotein A1 and totalcholesterol reached statistical significance with p-value lower than0.05, but % change was less than 10% between controls and cases, otherclinical values did not show any statistical significance. Thepredictive value of new lipidomic biomarkers was increased when theirlevels were expressed as distinct lipid-lipid ratios or lipid-clinicalratios (e.g. LDL-C or HDL-C).

Furthermore, to demonstrate improved diagnostic potential, logisticmodels were fitted in order to find different combinations of lipidsthat could separate cases and controls from each other. The lipids wereset as possible explanatory variables and model was selected usingstepwise method with different entry and stay significance levels. Themarkers with best diagnostic potential are listed in Table 9.

Importance of Detailed Molecular Lipid Analyses

Recent evolvement of mass spectrometry driven lipid analysis approacheshas made it possible to resolve complex lipidomes to their molecularlipid species level at high-throughput and quality required for analysesof clinical cohorts. As a result of the high sensitivity and selectivityof the methods, a lipidome-wide analysis of minute sample amounts hasbecome feasible. Present technologies are capable of identifying lipidswith different sum compositions, i.e. phosphatidylcholine (PC) 34:1, butmore important is the identification of molecular lipid species, e.g. PC16:0/18:1. In the latter analysis, information of the type of fattyacids and their positions attached to the glycerol backbone making upthe particular PC molecule is retrieved.

The seminal work of Shinzawa-Itoh and colleagues showed by highlysophisticated experiments that the oxygen transfer mechanism incytochrome c oxidase requires a specific phosphatidylglycerol molecularlipid with palmitate and vaccenate at the sn-1 and sn-2 positionsrespectively on the glycerol backbone (Shinzawa-Itoh K, Aoyama H,Muramoto K et al: Structures and physiological roles of 13 integrallipids of bovine heart cytochrome c oxidase. EMBO J. 2007,26:1713-1725). In line with other studies, this undoubtedly indicatesthat the lipid structure is an essential determinant of the biologicaleffect. Therefore, molecular lipidomics is an essential for biomarkerdiscovery. FIG. 1 illustrates the importance of molecular lipid data bycomparing the biomarker value of two PC and LacCer molecules inpredicting CVD mortality in the LURIC cohort. The data reveals thatwhile LacCer(d18:1/20:0) is a significant CVD predictor, LacCer(d18:1/18:16:0) has low biomarker potential. In addition, two PCmolecules PC (18:0/20:4) and PC (18:0/16:0) have even opposite effectson CVD complications. Thus, it is always necessary to identify andquantify all lipid species for lipid classes of interest including butnot limited to cholesterol esters, different phopsholipid classes,ceramides, cerebrosides (lactosylceramides, glycosylceramides), andgangliosides.

TABLE 3 Significant markers (lipid-lipid concentration ratios andlipid-clinical concentration ratios) for CVD patients undergoing statintreatment. Lipid names, p-values, % change, AUC, Sensitivity andSpecificity are presented. Percentage Measurement name P-value changeAUC Sensitivity Specificity Lipid-lipid concentration ratios IncreasedCE 19:1 (oxCE 682.6)/CE 20:5 0.01506 53.8 CE 19:1 (oxCE 682.6)/CE 20:40.01570 42.6 Cer(d18:1/16:0)/PC 18:1/20:4 0.01461 25.0Cer(d18:1/20:0)/PC 18:1/20:4 0.00899 23.8 CE 17:1/CE 20:5 0.01656 23.3SM (d18:1/15:0) (d18:1/14:1-OH)/SM 0.01678 20.6 (d18:1/23:0)(d18:1/22:1-OH) Cer(d18:1/16:0)/Cer(d18:1/24:0) 0.00268 20.4 CE 17:1/CE18:3 0.00557 18.4 CE 18:1/CE 18:3 0.00614 16.9 CE 16:0/CE 20:4 0.0042016.0 Decreased Cer(d18:1/22:0)/Cer(d18:1/24:1) 0.02889 −8.1 CE 14:0/CE17:1 0.01331 −9.7 CE 20:5/SM (d18:1/16:0) (d18:1/15:1- −10.5 0.59 60.960.0 OH) CE 14:0/CE 15:0 0.02897 −11.4 CE 14:0/Cer(d18:1/20:0) −13.80.62 61.2 60.9 Cer(d18:1/24:0)/Cer(d18:1/24:1) 0.00156 −15.2 0.67 65.761.5 CE 20:4/Cer(d18:1/20:0) 0.01809 −15.8 0.64 62.9 60.0 CE14:0/Cer(d18:1/16:0) 0.02068 −16.7 0.63 64.2 60.9 CE20:4/Cer(d18:1/16:0) 0.00168 −18.5 0.66 65.7 60.0 CE20:5/Cer(d18:1/20:0) −18.6 0.61 60.9 66.2 CE 20:5/Cer(d18:1/24:1) −19.70.62 60.9 60.0 CE 20:5/Cer(d18:1/16:0) 0.00991 −22.7 CE20:5/Cer(d18:1/26:1) 0.00270 −24.5 0.68 67.2 61.8 Lipid-clinicalconcentration ratios Increased CE 19:1 (oxCE 682.6)/LDL cholesterol0.00743 35.7 0.63 61.7 62.7 Cer(d18:1/16:0)/HDL cholesterol 0.00966 23.00.64 62.9 63.1 Cer(d18:1/16:0)/apolipoprotein B 0.00305 17.7 0.64 62.961.5 Decreased Cer(d18:1/24:0)/supersensitive C-reactive −27.5 0.66 63.664.4 protein Gb3(d18:1/18:0)/supersensitive C-reactive −36.4 0.72 73.865.5 protein Cer(d18:1/18:0)/supersensitive C-reactive 0.02424 −42.60.71 72.9 60.0 protein GlcCer(d18:1/22:0)/supersensitive C- 0.03076−42.9 0.72 77.1 60.0 reactive protein LacCer(d18:1/24:1)/supersensitiveC- 0.03423 −43.1 0.72 77.1 60.0 reactive protein PC16:0/18:1/supersensitive C-reactive 0.02417 −43.2 0.71 71.4 64.6 proteinCer(d18:1/24:1)/supersensitive C-reactive 0.02327 −43.5 0.71 72.9 61.5protein GlcCer(d18:1/20:0)/supersensitive C- 0.02590 −43.9 0.72 79.160.9 reactive protein Cer(d18:1/16:0)/supersensitive C-reactive 0.02606−44.3 0.71 74.3 60.0 protein GlcCer(d18:1/24:1)/supersensitive C-0.02371 −44.4 0.71 78.6 63.1 reactive proteinLacCer(d18:1/24:0)/supersensitive C- 0.02599 −44.5 0.72 74.1 66.7reactive protein Gb3(d18:1/24:0)/supersensitive C-reactive 0.01784 −45.00.74 75.0 69.4 protein PC 18:0/20:3/supersensitive C-reactive 0.01232−45.1 0.74 77.6 62.5 protein CE 16:0/supersensitive C-reactive 0.01659−46.4 0.73 78.6 60.0 protein SM (d18:1/18:0)/supersensitive C-reactive0.01375 −46.5 0.73 78.3 61.5 protein CE 20:3/supersensitive C-reactive0.00475 −46.5 0.73 75.4 62.5 protein CE 18:1/supersensitive C-reactive0.01116 −46.6 0.73 74.3 61.5 protein Cer(d18:1/22:0)/supersensitiveC-reactive 0.01273 −46.7 0.72 75.7 60.0 proteinLacCer(d18:1/16:0)/supersensitive C- 0.02583 −46.7 0.72 75.7 60.9reactive protein GlcCer(d18:1/16:0)/supersensitive C- 0.01003 −47.4 0.7274.3 60.9 reactive protein GlcCer(d18:1/24:0)/supersensitive C- 0.01065−47.9 0.72 75.7 66.2 reactive protein SM (d18:1/18:1)/supersensitiveC-reactive 0.01020 −47.9 0.73 77.1 61.5 proteinGlcCer(d18:1/18:0)/supersensitive C- 0.00841 −48.3 0.73 75.7 62.5reactive protein PC O-18:0/18:2-alkyl/supersensitive C- 0.02112 −48.30.71 74.6 66.1 reactive protein SM (d18:1/16:1) (d18:1/15:2- 0.00678−48.8 0.72 77.1 60.0 OH)/supersensitive C-reactive proteinCer(d18:1/20:0)/supersensitive C-reactive 0.01211 −49.3 0.72 72.9 64.6protein CE 14:0/supersensitive C-reactive 0.00711 −49.3 0.71 74.6 62.5protein SM (d18:1/23:0) (d18:1/22:1- 0.00848 −49.3 0.74 80.0 60.0OH)/supersensitive C-reactive protein Gb3(d18:1/16:0)/supersensitiveC-reactive 0.01307 −49.4 0.72 77.1 62.5 protein SM (d18:1/14:0)(d18:1/13:1- 0.00643 −49.6 0.72 75.7 63.1 OH)/supersensitive C-reactiveprotein PC 16:0/16:0/supersensitive C-reactive 0.01232 −49.8 0.71 73.962.5 protein PC 18:0/18:2/supersensitive C-reactive 0.00652 −49.9 0.7278.6 60.0 protein PC 18:1/20:4/supersensitive C-reactive 0.01737 −49.90.74 79.0 61.4 protein SM (d18:1/24:0) (d18:1/23:1- 0.00971 −50.3 0.7481.4 64.6 OH)/supersensitive C-reactive protein CE 18:3/supersensitiveC-reactive 0.00367 −50.7 0.73 77.1 63.1 proteinCer(d18:1/24:0)/supersensitive C-reactive 0.00487 −51.1 0.72 75.7 61.5protein CE 18:2/supersensitive C-reactive 0.00505 −51.2 0.73 78.6 60.0protein Cer(d18:1/26:1)/supersensitive C-reactive 0.01133 −51.6 0.7172.9 65.5 protein DAG 16:0/18:1/supersensitive C-reactive 0.00501 −52.10.71 76.5 62.9 protein PC 18:1/18:2/supersensitive C-reactive 0.00743−52.1 0.71 75.7 61.5 protein LPC 16:0/supersensitive C-reactive 0.00829−52.5 0.73 80.0 60.9 protein CE 20:4/supersensitive C-reactive 0.00302−52.9 0.74 78.6 63.1 protein PC-0 16:0/24-alkyl/CRP 0.01217 −53.3 0.7685.2 63.5 PC O-16:0/18:2-alkyl/supersensitive C- 0.00961 −53.9 0.74 79.361.4 reactive protein PC 16:0/22:6/supersensitive C-reactive 0.00247−55.3 0.73 75.7 62.5 protein PC 18:0/22:6/supersensitive C-reactive0.00464 −55.9 0.76 76.3 60.0 protein CE 20:5/supersensitive C-reactive0.00193 −59.9 0.71 68.1 60.0 protein

TABLE 4 Significant markers for CVD patients undergoing statin treatmentand not suffering from diabetes. Lipid names, p-values, % change, AUC,Sensitivity and Specificity values are presented. Table 4a shows asignificant lipid marker, Table 4b shows lipid-lipid concentration ratiomarkers and Table 4c shows lipid-clinical concentration ratio markersPercentage Measurement name P-value change AUC Sensitivity Specificity4a) Significant lipid marker for CVD patients undergoing statintreatment and not suffering from diabetes. Lipid Increased SM(d18:1/17:0) (d18:1/16:1-OH) 0.03343 19.0 4b) Significant lipid-lipidconcentration ratio markers for CVD patients undergoing statin treatmentand not suffering from diabetes. Lipid-lipid concentration ratiosIncreased Cer(d18:1/26:1)/SM (d18:1/24:0) 0.00273 41.4 0.66 64.4 67.3(d18:1/23:1-OH) CE 16:1/SM (d18:1/24:0) (d18:1/23:1-OH) 39.0 0.64 64.360.0 PC 16:0/16:1/PC O-16:0/20:4-alkyl 36.3 0.65 60.4 62.7 PC16:0/16:1/PC 18:2/18:2 31.8 0.64 63.8 62.3 CE 16:1/CE 20:5 0.00374 31.60.64 66.7 60.0 PC 16:0/16:1/PC 18:1/20:4 31.2 0.64 65.0 61.1 PC18:0/18:1/PC 18:2/18:2 0.00984 31.0 0.65 65.6 64.9 Cer(d18:1/18:0)/PCO-16:0/20:4-alkyl 0.00650 30.2 Cer(d18:1/20:0)/PC 18:2/18:2 0.00793 28.40.65 65.6 68.4 Cer(d18:1/20:0)/PC O-16:0/20:4-alkyl 0.00700 27.3 CE16:1/CE 20:4 26.5 0.65 67.1 61.5 Cer(d18:1/24:1)/PC 18:2/18:2 0.0131826.4 0.65 73.8 63.2 Cer(d18:1/24:1)/PC 18:1/20:4 0.00455 26.4 0.65 62.963.2 CE 16:1/PC 18:2/18:2 25.3 0.66 62.3 61.4 CE 16:1/CE 18:3 0.0002824.5 0.67 62.9 60.0 Cer(d18:1/18:0)/PC O-16:0/18:2-alkyl 24.2 0.65 65.563.2 CE 17:1/CE 20:4 0.00196 23.8 PC 16:0/18:1/PC 18:1/20:4 0.00737 23.7PC 16:0/18:1/PC O-16:0/20:4-alkyl 23.3 0.66 66.7 61.5 CE 16:1/PCO-16:0/20:4-alkyl 23.2 0.65 64.8 61.5 PC 18:1/18:1/PC 18:1/20:4 0.0113122.6 0.65 66.1 61.4 SM (d18:1/16:1) (d18:1/15:2-OH)/SM 21.6 0.64 65.760.0 (d18:1/24:0) (d18:1/23:1-OH) CE 18:1/CE 20:4 0.00776 20.4Cer(d18:1/18:0)/Cer(d18:1/24:0) 0.01375 20.4 0.65 65.7 63.1Cer(d18:1/24:1)/PC O-16:0/18:2-alkyl 0.01544 18.8 0.65 67.2 61.4 PC18:0/18:1/PC 18:0/20:3 0.00714 18.8 Cer(d18:1/26:1)/PC 16:0/22:6 18.50.69 69.5 61.1 CE 16:1/PC 18:1/20:4 16.7 0.65 67.7 61.4Cer(d18:1/26:1)/PC 18:0/18:2 16.6 0.65 62.7 60.0 CE 16:1/Cer(d18:1/24:0)16.5 0.65 65.7 60.0 Cer(d18:1/26:1)/PC 18:1/18:2 16.2 0.64 64.4 60.0Cer(d18:1/26:1)/PC 16:0/18:2 0.01662 14.6 0.64 61.0 65.5 PC 18:1/18:2/PC18:2/18:2 0.00938 13.9 0.66 70.5 61.4 PC 16:0/18:2/PC 18:2/18:2 12.80.64 65.6 61.4 Decreased CE 14:0/CE 16:1 0.00317 −14.9 CE 18:3/PC16:0/18:1 0.00429 −16.6 0.65 60.0 66.2 CE 14:0/PC 18:0/18:1 0.00333−17.1 Cer(d18:1/24:0)/Cer(d18:1/26:1) 0.00253 −18.6 0.66 62.7 61.8 CE20:4/PC 18:0/18:1 0.00578 −18.7 0.63 60.9 60.0 Gb3(d18:1/22:0)/PC16:0/18:1 0.00732 −18.9 CE 18:3/PC 18:0/18:1 0.00128 −19.2 CE 20:5/PC16:0/16:1 −20.1 0.64 65.1 66.1 CE 20:4/Cer(d18:1/26:1) 0.00519 −21.40.65 72.9 60.0 CE 18:3/PC 16:0/16:1 0.00529 −21.4 0.64 65.6 61.0 CE18:3/Cer(d18:1/26:1) 0.00817 −25.2 0.66 71.2 60.0 LPC 16:0/SM(d18:1/17:0) (d18:1/16:1-OH) 0.00816 −25.3 Gb3(d18:1/22:0)/SM(d18:1/17:0) 0.00370 −27.6 (d18:1/16:1-OH) CE 20:5/CE 22:2 −29.4 0.6462.3 77.4 4c) Significant lipid - clinical concentration ratio markersfor CVD patients undergoing statin treatement and not suffering fromdiabetes. Lipid-clinical concentration ratios Increased SM (d18:1/17:0)(d18:1/16:1-OH)/HDL 0.00589 34.2 cholesterol CE 22:2/HDL cholesterol0.02679 32.0 0.62 63.0 60.4 SM (d18:1/17:0) (d18:1/16:1- 0.00844 31.9OH)/apolipoprotein A-I PC 16:0/16:1/apolipoprotein A-I 0.04344 28.5 0.6364.1 61.0 CE 22:2/apolipoprotein A-I 0.03811 28.1 0.61 63.0 64.2 PC16:0/16:1/HDL cholesterol 27.2 0.62 65.6 61.0 Cer(d18:1/26:1)/HDLcholesterol 0.00274 27.2 Cer(d18:1/26:1)/apolipoprotein A-I 0.00203 25.60.65 61.0 60.0 PC 18:0/18:1/apolipoprotein A-I 0.01304 25.5 PC18:0/18:1/total cholesterol 0.00849 24.6 PC 16:0/18:1/apolipoprotein A-I0.01425 22.8 PC 16:0/18:1/apolipoprotein A-I 0.01425 22.8 0.64 65.7 60.0Cer(d18:1/26:1)/LDL cholesterol 0.01464 22.6 PC 16:0/18:1/totalcholesterol 0.00680 21.6 PC 18:1/18:1/apolipoprotein A-I 0.01499 21.4Cer(d18:1/26:1)/total cholesterol 0.00212 21.1 0.67 66.1 60.0 CE15:0/HDL cholesterol 0.04918 20.4 0.62 63.5 61.0 PC 18:1/18:1/HDLcholesterol 19.8 0.63 61.8 61.3 CE 15:0/apolipoprotein A-I 19.7 0.6160.3 64.4 PC 18:1/18:1/total cholesterol 0.01779 19.7 CE 17:1/HDLcholesterol 0.03189 19.7 0.61 62.5 60.0 CE 17:1/apolipoprotein A-I0.03710 18.6 0.61 60.9 61.7 Cer(d18:1/26:1)/apolipoprotein B 0.0143118.0 0.66 62.7 60.0 PC 16:0/18:1/LDL cholesterol 15.4 0.64 67.1 60.0 CE22:6/HDL cholesterol 13.5 0.58 64.3 61.5 PC 16:0/18:2/LDL cholesterol5.2 0.59 62.9 60.0 Decreased SM (d18:1/24:0) (d18:1/23:1- −14.1 0.6061.4 60.0 OH)/triglycerides CE 19:1 (oxCE 682.6)/supersensitive C- −32.90.67 71.7 61.0 reactive protein PC 17:0/18:2/supersensitive C-reactive−39.7 0.71 71.2 67.3 protein SM (d18:1/17:0) (d18:1/16:1- −43.4 0.6872.4 60.7 OH)/supersensitive C-reactive proteinGb3(d18:1/24:1)/supersensitive C-reactive 0.02892 −44.3 0.73 79.7 60.9protein Gb3(d18:1/22:0)/supersensitive C-reactive 0.01805 −46.6 0.7577.6 64.4 protein SM (d18:1/15:0) (d18:1/14:1- 0.01189 −48.1 0.71 75.767.7 OH)/supersensitive C-reactive protein SM (d18:1/23:1) (d18:1/22:2-0.01156 −48.5 0.74 78.0 60.7 OH)/supersensitive C-reactive protein CE22:6/supersensitive C-reactive 0.01571 −48.7 0.72 77.1 60.0 protein CE15:0/supersensitive C-reactive 0.01649 −48.8 0.69 73.0 61.0 protein SM(d18:1/16:0) (d18:1/15:1- 0.00532 −48.9 0.73 80.0 60.0OH)/supersensitive C-reactive protein PC 16:0/18:2/supersensitiveC-reactive 0.00493 −50.1 0.72 75.7 60.0 protein SM (d18:1/24:1)(d18:1/23:2- 0.00411 −51.7 0.73 75.7 62.5 OH)/supersensitive C-reactiveprotein PC 18:2/18:2/supersensitive C-reactive 0.01127 −57.7 0.74 77.061.4 protein

TABLE 5 Significant markers for CVD patients undergoing statin treatmentand having diabetes. Marker names, p-values, percentage change, AUC,Specificity and Sensitivity are presented. Table 5a shows significantlipid markers, Table 5b shows significant lipid-lipid concentrationratio markers and Table 5c shows significant lipid-clinicalconcentration ratio markers Percentage Measurement name P-value changeAUC Sensitivity Specificity 5a) Significant lipid markers for CVDpatients undergoing statin treatment and having diabetes. LipidsIncreased CE 19:1 (oxCE 682.6) 0.02810 41.6 0.65 68.4 66.7 CE 20:0 40.10.61 60.0 61.5 Decreased PC 18:2/18:2 −4.6 0.59 61.1 61.5 CE 22:6 −4.60.60 60.9 62.1 Cer(d18:1/24:0) 0.02322 −13.4 0.67 70.8 65.5 PC 18:0/22:6−15.3 0.62 64.8 63.6 PC P-18:0/20:4 0.01681 −15.8 0.67 68.4 60.9 CE 18:10.01154 −16.3 0.63 62.5 62.1 PC O-16:0/20:4-alkyl 0.01000 −17.3 LPC 16:00.02481 −17.6 DAG 16:0/18:2 −20.1 0.64 64.0 60.9 CE 20:4 0.00295 −23.10.75 79.7 62.1 PC 18:1/20:4 0.00057 −24.5 0.72 63.5 61.5 PC 16:0/16:10.04569 −25.8 0.65 64.2 66.7 DAG 16:0/18:1 0.01553 −27.1 0.68 71.9 60.7CE 16:1 0.00304 −32.0 0.70 68.8 69.0 CE 18:3 0.00011 −32.4 0.71 70.365.5 CE 20:5 0.00043 −34.7 0.72 70.3 65.5 5b) Significant lipid-lipidconcentration ratio markers for CVD patients undergoing statin treatmentand having diabetes. Lipid-lipid concentration ratios IncreasedCer(d18:1/16:0)/DAG 16:0/18:1 89.1 0.72 67.2 60.7 Cer(d18:1/16:0)/PC18:0/20:3 0.00075 50.2 0.73 73.4 62.1 Cer(d18:1/20:0)/PC 18:0/20:30.00082 47.3 0.72 70.3 62.1 PC 16:0/16:0/PC 18:1/20:4 0.00022 42.5 0.7776.9 61.5 CE 15:0/CE 18:3 0.00105 41.1 0.72 71.4 63.0 CE 15:0/CE 16:10.00053 40.3 0.73 75.0 66.7 PC 17:0/18:2/PC 18:1/20:4 0.00226 39.3 0.7881.6 63.6 PC 16:0/18:2/PC 18:1/20:4 0.00037 33.0 0.78 76.9 69.2 PC18:0/18:2/PC 18:1/20:4 0.00547 27.4 0.73 76.9 61.5 Decreased CE 18:1/SM(d18:1/24:1) 0.00183 −22.9 0.72 67.2 65.5 (d18:1/23:2-OH) PCP-18:0/20:4/SM (d18:1/24:1) −24.2 0.76 77.2 69.6 (d18:1/23:2-OH) CE18:1/Cer(d18:1/16:0) 0.00037 −24.2 CE 16:1/CE 17:1 0.00074 −24.6 0.7570.0 63.0 CE 18:3/PC 18:0/18:2 0.00044 −24.7 0.72 75.0 62.1 PC18:0/20:3/SM (d18:1/16:0) −25.4 0.72 73.4 62.1 (d18:1/15:1-OH) CE18:3/GlcCer(d18:1/16:0) −26.0 0.72 67.2 62.1 CE 18:3/PC 16:0/18:20.00013 −26.5 0.74 81.3 62.1 CE 14:0/Gb3(d18:1/16:0) −28.2 0.73 79.769.0 PC 18:1/20:4/SM (d18:1/16:0) 0.00151 −28.2 0.74 78.8 61.5(d18:1/15:1-OH) CE 18:3/SM (d18:1/14:0) −28.5 0.73 73.0 62.1(d18:1/13:1-OH) CE 18:3/PE 18:0/18:2 0.00036 −28.8 0.74 80.8 63.6 PC18:1/20:4/PC O-18:0/18:2-alkyl 0.00090 −28.8 0.77 80.0 60.9 CE 20:4/SM(d18:1/24:1) 0.00082 −29.4 0.74 71.9 62.1 (d18:1/23:2-OH) CE16:1/Gb3(d18:1/16:0) −30.2 0.73 76.6 62.1 CE 20:4/PC 17:0/18:2 0.00095−30.4 PC 18:1/20:4/SM (d18:1/24:1) 0.00122 −30.5 0.77 76.9 61.5(d18:1/23:2-OH) CE 18:3/SM (d18:1/16:1) −30.6 0.73 78.1 62.1(d18:1/15:2-OH) CE 16:1/SM (d18:1/16:0) −31.2 0.73 73.4 62.1(d18:1/15:1-OH) CE 18:3/Gb3(d18:1/22:0) −31.2 0.72 69.5 62.1 CE18:3/LacCer(d18:1/16:0) 0.00079 −31.8 0.73 71.9 65.5 CE 18:3/SM(d18:1/16:0) 0.00112 −32.1 0.75 75.0 62.1 (d18:1/15:1-OH) PC18:1/20:4/SM (d18:1/15:0) −32.6 0.72 69.4 61.5 (d18:1/14:1-OH) CE18:3/Gb3(d18:1/16:0) −32.8 0.76 81.3 65.5 CE 18:3/SM (d18:1/18:0) −33.20.72 65.6 75.9 CE 16:1/SM (d18:1/24:1) −35.1 0.74 71.9 62.1(d18:1/23:2-OH) CE 16:1/Cer(d18:1/16:0) 0.00110 −35.3 0.76 76.6 69.0 CE20:5/LacCer(d18:1/16:0) 0.00031 −35.7 0.71 65.6 65.5 CE 18:3/SM(d18:1/24:1) 0.00039 −36.6 0.75 75.0 62.1 (d18:1/23:2-OH) CE 18:3/SM(d18:1/15:0) −36.9 0.73 70.5 62.1 (d18:1/14:1-OH) CE20:5/Gb3(d18:1/16:0) 0.00052 −38.9 0.72 70.3 62.1 CE 20:5/SM(d18:1/24:1) 0.00047 −39.1 0.74 71.9 65.5 (d18:1/23:2-OH) 5c)Significant lipid-clinical concentration ratio markers for CVD patientsundergoing statin treatment and having diabetes. Lipid-clinicalconcentration ratios Increased CE 20:0/apolipoprotein A-I 0.02742 70.80.66 63.6 61.5 CE 20:0/total cholesterol 0.03377 51.9 0.64 63.6 61.5 SM(d18:1/24:1) (d18:1/23:2- 0.00512 29.2 0.69 73.4 62.1 OH)/apolipoproteinA-I SM (d18:1/24:1) (d18:1/23:2- 0.00836 28.9 OH)/HDL cholesterol SM(d18:1/24:1) (d18:1/23:2- 0.01047 24.6 OH)/total cholesterolGb3(d18:1/16:0)/HDL cholesterol 0.02324 23.7 0.65 60.0 62.1 SM(d18:1/16:0) (d18:1/15:1- 0.01268 22.9 OH)/apolipoprotein A-I PCO-18:0/18:2- 0.02531 22.6 0.64 66.7 60.0 alkyl/apolipoprotein A-IGb3(d18:1/16:0)/apolipoprotein 0.00747 22.4 0.68 61.5 62.1 A-I SM(d18:1/24:1) (d18:1/23:2- 22.0 0.64 60.9 62.1 OH)/triglycerides SM(d18:1/15:0) (d18:1/14:1- 21.8 0.64 68.9 65.5 OH)/triglyceridesGb3(d18:1/16:0)/triglycerides 21.1 0.64 63.1 62.1 PC17:0/18:2/triglycerides 20.6 0.64 66.1 60.9 Gb3(d18:1/16:0)/totalcholesterol 0.00943 18.7 0.68 66.2 65.5 Decreased PCP-18:0/20:4/apolipoprotein B −12.6 0.67 64.9 60.9 CE 20:4/apolipoproteinA-I −13.1 0.64 60.9 62.1 CE 14:0/HDL cholesterol −15.3 0.64 64.1 65.5 PC18:1/20:4/HDL cholesterol −15.9 0.64 61.5 61.5 CE 20:4/total cholesterol0.02956 −16.6 0.67 64.1 62.1 PC 16:0/16:1/apolipoprotein B −18.2 0.6560.4 66.7 PC 18:1/20:4/total cholesterol 0.00869 −18.5 0.70 76.9 65.4 CE20:4/apolipoprotein B 0.00339 −20.4 0.70 65.6 62.1 PC18:0/20:3/apolipoprotein B 0.00280 −21.4 0.68 60.9 62.1 DAG16:0/18:1/apolipoprotein B 0.03441 −21.9 0.66 62.5 60.7 CE 20:3/LDLcholesterol 0.01949 −22.1 0.66 67.2 65.5 PC 18:1/20:4/apolipoprotein B0.00085 −22.9 0.74 82.7 61.5 PC 18:0/20:3/LDL cholesterol 0.02201 −23.50.67 60.9 62.1 CE 20:4/LDL cholesterol 0.00523 −25.5 0.69 68.8 65.5 CE20:5/total cholesterol 0.00307 −26.8 PC 18:1/20:4/LDL cholesterol0.00369 −28.5 0.71 69.2 65.4 PC P-16:0/18:2/supersensitive C- −35.1 0.7062.3 63.6 reactive protein DAG 16:0/18:2/supersensitive C- −35.4 0.7572.0 60.9 reactive protein CE 19:2 (oxCE −40.0 0.72 62.3 63.6680.6)/supersensitive C-reactive protein PC P-18:0/20:4/supersensitiveC- −40.7 0.72 66.7 60.9 reactive protein

TABLE 6 The preferred embodiment markers for CVD patients undergoingstatin treatment. Percentage Measurement P-value change AUC SensitivitySpecificity Lipid-lipid concentration ratio Increased CE 19:1 (oxCE682.6)/CE 20:5 0.01506 53.8 CE 19:1 (oxCE 682.6)/CE 20:4 0.01570 42.6Cer(d18:1/16:0)/PC 18:1/20:4 0.01461 25.0Cer(d18:1/16:0)/Cer(d18:1/24:0) 0.00268 20.4 DecreasedCer(d18:1/24:0)/Cer(d18:1/24:1) 0.00062 −16.9 0.70 74.5 60.9 CE20:5/Cer(d18:1/26:1) 0.00270 −24.5 0.68 67.2 61.8 Lipid-clinicalconcentration ratios Increased Cer(d18:1/16:0)/apolipoprotein B 0.0030517.7 0.64 62.9 61.5 Decreased Cer(d18:1/24:0)/supersensitive C- −27.50.66 63.6 64.4 reactive protein PC 18:0/22:6/supersensitive C- −28.80.65 62.8 65.8 reactive protein CE 18:3/supersensitive C-reactive0.00367 −50.7 0.73 77.1 63.1 protein LPC 16:0/supersensitive C-reactive0.00829 −52.5 0.73 80.0 60.9 protein PC-0 16:0/24-alkyl/CRP 0.01217−53.3 0.76 85.2 63.5 PC 16:0/22:6/supersensitive C- 0.00247 −55.3 0.7375.7 62.5 reactive protein CE 20:5/supersensitive C-reactive 0.00193−59.9 0.71 68.1 60.0 protein

TABLE 7 The preferred embodiment markers from CVD patients undergoingstatin treatment and not having diabetes. Percentage Measurement nameP-value change AUC Sensitivity Specificity Lipid Increased SM(d18:1/17:0) (d18:1/16:1-OH) 0.03343 19.0 Lipid-lipid concentrationratios Increased Cer(d18:1/26:1)/SM (d18:1/24:0) 0.00273 41.4 0.66 64.467.3 (d18:1/23:1-OH) PC 16:0/16:1/PC 18:1/20:4 31.2 0.64 65.0 61.1Decreased Gb3(d18:1/22:0)/PC 16:0/18:1 0.00732 −18.9 CE20:4/Cer(d18:1/26:1) 0.00519 −21.4 0.65 72.9 60.0 CE 20:5/CE 22:2 −29.40.64 62.3 77.4 Lipid-clinical concentration ratios Increased SM(d18:1/17:0) (d18:1/16:1- 0.00589 34.2 OH)/HDL cholesterol CE 22:2/HDLcholesterol 0.02679 32.0 0.62 63.0 60.4 PC 16:0/16:1/apolipoprotein A-I0.04344 28.5 0.63 64.1 61.0 Decreased Gb3(d18:1/22:0)/supersensitive C-0.01805 −46.6 0.75 77.6 64.4 reactive protein CE 22:6/supersensitiveC-reactive 0.01571 −48.7 0.72 77.1 60.0 protein PC18:2/18:2/supersensitive C- 0.01127 −57.7 0.74 77.0 61.4 reactiveprotein

TABLE 8 The preferred embodiment markers from CVD patients undergoingstatin treatment and having diabetes. Percentage Measurement nameP-value change AUC Sensitivity Specificity Lipids Increased CE 19:1(oxCE 682.6) 0.02810 41.6 0.65 68.4 66.7 CE 20:0 40.1 0.61 60.0 61.5Decreased CE 16:1 0.00304 −32.0 0.70 68.8 69.0 CE 18:3 0.00011 −32.40.71 70.3 65.5 CE 20:5 0.00043 −34.7 0.72 70.3 65.5 Lipid-lipidconcentration ratios Increased Cer(d18:1/16:0)/DAG 16:0/18:1 89.1 0.7267.2 60.7 Cer(d18:1/16:0)/PC 18:0/20:3 0.00075 50.2 0.73 73.4 62.1 PC17:0/18:2/PC 18:1/20:4 0.00226 39.3 0.78 81.6 63.6 PC 16:0/18:2/PC18:1/20:4 0.00037 33.0 0.78 76.9 69.2 Lipid-clinical concentrationratios Increased CE 20:0/apolipoprotein A-I 0.02742 70.8 0.66 63.6 61.5Decreased DAG 16:0/18:2/supersensitive C- −35.4 0.75 72.0 60.9 reactiveprotein

The preferred lipid molecules of the invention were selected as follows:a) it was likely to be biologically meaningful, b) it preferably belongsto a family of lipids that are behaving similarly, c) it is expressed inmeaningful & measurable concentrations, d) it has very significantp-value or good AUC-value (>0.65) and for most also the %-change issubstantial (>20%), and e) it appeared significant in different tests

TABLE 9 Lipid markers generated with logistic modeling. CVD high riskmarkers for CVD high risk markers for CVD patients on statin CVDpatients on statin treatment and not having diabetes treatment andhaving diabetes Increased Increased Cer 18:1/24:0 Cer 18:1/16:0 PC16:0/22:6 CE 16:0 GlcCer 18:1/24:1 PC 16:0/18:2 CE 18:3 Cer 18:1/20:0 SM18:1/24:1 Decreased Decreased GlcCer 18:1/18:0 CE 20:5 Cer 18:1/16:0 Cer18:1/18:0 Cer 18:1/24:1 CE 20:3 PC 18:0/18:1 PC 16:0/16:0 Cer 18:1/22:0PC 18:1/18:2 CE 16:1 CE 18:1 CE 20:4

Lipidomic analysis proved to be efficient in identifying novel plasmabiomarkers for CVD complications.

Molecular lipid to molecular lipid ratio could be an important indicatorof cellular lipid metabolism including e.g., enzyme activities in thelipid metabolism pathways. Thus, these ratios may provide moreinformation as the absolute plasma concentrations of the molecularlipids alone. As the absolute molecular lipid plasma concentrationdifferences in general between healthy individuals and atheroscleroticpatients seem to be between 30-70%, it might be reasonable to calculateand use different ratios instead of absolute concentrations only. Aslipoprotein particles (e.g. LDL, HDL, and VLDL) are serving as carriersfor most of the lipids in the blood stream it is appropriate to relatemolecular lipid concentrations to lipoprotein data. Thus, the molecularlipid to HDL-cholesterol, LDL-cholesterol, apolipoprotein A-I andapolipoprotein B ratios were calculated. In fact, a number of ratiosbetween the concentrations of different molecular lipids outperformedabsolute plasma concentrations as disease biomarkers in CVD patients.

As the detected lipids are carried in the lipoprotein particles (LDL,VLDL and HDL) it is obvious that the corresponding lipoprotein fractionconcentrations will even improve the prediction potential of molecularlipids from the results of the present study in total serum/plasmasamples.

The lipid lowering drug efficiency measurements have so far been basedon LDL-C and HDL-C assays. As the inventors have herein observed morepotential biomarkers that predict the development of high-risk CVDcomplications better than these classical analyses, future drugefficiency profiling should be based on new sensitive and specificbiomarkers that are more directly related to the risk of severeCVD-related complications rather than to LDL-C.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, numerous equivalents to thespecific embodiments described herein both in the Examples in the bodyof the entire patent description. Such equivalents are considered to bewithin the scope of this invention and are covered by the followingclaims.

The invention claimed is:
 1. A method for determining whether a subjectundergoing statin treatment is at risk to develop one or morecardiovascular disease (CVD) complications, comprising a. determining ina sample from said subject one or more lipid-lipid concentrationratio(s), wherein (an) increased or decreased lipid-lipid concentrationratio(s) in said sample, when compared to a control sample, is (are)indicative of said subject having an increased risk of developing one ormore CVD complications, wherein the one or more lipid-lipidconcentration ratio(s) whose increase(s) is (are) compared to thecontrol is (are) selected from: Cer(d18:1/16:0)/Cer(d18:1/24:0),Cer(d18:1/16:0)/PC 18:1/20:4; and wherein the one or more lipid-lipidconcentration ratio(s) whose decrease(s) is (are) compared to thecontrol is Cer(d18:1/24:0)/Cer(d18:1/24:1); or b. determining in asample from said subject one or more lipid-clinical concentrationratio(s), wherein (an) increased or decreased lipid-clinicalconcentration ratio(s) in said sample, when compared to a controlsample, is (are) indicative of said subject having an increased risk ofdeveloping one or more CVD complications, wherein the one or morelipid-clinical concentration ratio(s) whose increase(s) is (are)compared to the control is (are) selected from:Cer(d18:1/16:0)/apolipoprotein B; and wherein the one or morelipid-clinical concentration ratio(s) whose decrease(s) is (are)compared to the control is Cer(d18:1/24:0)/supersensitive C-reactiveprotein.
 2. The method of claim 1, wherein determining the lipid-lipidconcentration ratio(s) or the lipid-clinical concentration ratio(s) isdone using mass spectrometry.
 3. The method of claim 1, comprisingdetermining at least 2, at least 3, at least 4, at least 5, at least 6,at least 7, or at least 8 lipid-lipid concentration ratios orlipid-clinical concentration ratios, respectively, or combinationsthereof.
 4. The method of claim 1, wherein a. said CVD is characterizedby coronary artery disease, peripheral artery disease, a stroke and/orCVD death; and/or b. said CVD is atherosclerosis-induced; and/or c. saidsubject has atherosclerosis; or d. said subject does not haveatherosclerosis.
 5. The method of claim 1, wherein a. the method furthercomprises determining the serum or plasma level of total cholesterol,low-density lipoprotein cholesterol (LDL-C), high-density lipoproteincholesterol (HDL-C), Apolipoprotein B (ApoB) and/or Apolipoprotein C-III(ApoC-III) in said sample; and/or b. the subject does not have elevatedserum or plasma levels of one or more of total cholesterol, low-densitylipoprotein cholesterol (LDL-C), Apolipoprotein C-III (ApoC-III) orApolipoprotein B (ApoB), or a decreased serum level of HDL-cholesterol(HDL-C).
 6. The method of claim 1, wherein a. the sample is blood,plasma, serum, urine or tissue, or a lipoprotein fraction thereof and/orb. the lipid-lipid concentration ratio(s) or the lipid-clinicalconcentration ratio(s) is (are) determined by using mass spectrometry,nuclear magnetic resonance spectroscopy, fluorescence spectroscopy ordual polarisation interferometry, a high performance separation method,an immunoassay and/or with a binding moiety capable of specificallybinding the analyte.
 7. The method of claim 1, wherein the one or moreCVD complications are selected from CVD death and acute myocardialinfarction (AMI).
 8. The method of claim 1, wherein the subject is atrisk to develop or has suffered from one or more CVD complications. 9.The method of claim 1, wherein the control sample is from (a) CADpatient(s) or a group of CAD patients, wherein the CAD patient(s) orgroup of CAD patients has/have no history of major CVD events and is/areundergoing statin treatment; wherein the control sample is blood,plasma, serum, urine or tissue, or a lipoprotein fraction thereof. 10.The method of claim 1, wherein the sample is blood, serum, or plasma andthe one or more lipid-lipid concentration ratio(s) or the one or morelipid-clinical concentration ratio(s) is (are) determined by using massspectrometry.
 11. The method of claim 10, further comprising a step ofobtaining the sample from the subject.
 12. The method of claim 10,further comprising a step of extracting lipids from the blood, serum, orplasma sample.
 13. The method of claim 10, wherein the one or more CVDcomplications are selected from CVD death and acute myocardialinfarction (AMI).
 14. The method of claim 11, wherein the one or moreCVD complications are selected from CVD death and acute myocardialinfarction (AMI).
 15. The method of claim 12, wherein the one or moreCVD complications are selected from CVD death and acute myocardialinfarction (AMI).